Pac-1 combination therapy

ABSTRACT

Compositions and methods for the induction of cancer cell death. The compositions and methods of using them include use of compositions in therapy for the treatment of cancer and for the selective induction of apoptosis in cancer cells. The drug combinations described herein can be synergistic and can have lower neurotoxicity effects than the same amounts of other compounds and combinations of compounds and can be effective when a particular cancer has become resistant to previously administered therapies.

RELATED APPLICATIONS

This application is a divisional application of U.S. patent applicationSer. No. 15/579,689 filed Dec. 5, 2017, which is a National Stage filingunder 35 U.S.C. § 371 of International Application No. PCT/US2016/036063filed Jun. 6, 2016, which claims priority under 35 U.S.C. § 119(e) toU.S. Provisional Patent Application Nos. 62/171,882 filed Jun. 5, 2015and 62/345,629 filed Jun. 3, 2016, which applications are incorporatedherein by reference.

BACKGROUND OF THE INVENTION

Apoptosis, or programmed cell death, plays a central role in thedevelopment and homeostasis of all multicellular organisms. A frequenthallmark of cancer is resistance to natural apoptotic signals. Dependingon the cancer type, this resistance can be due to up- or down-regulationof key proteins in the apoptotic cascade. The resistance can also be dueto mutations in genes encoding these proteins. These changes can occurin both the intrinsic apoptotic pathway, which funnels through themitochondria and caspase-9, and the extrinsic apoptotic pathway, whichinvolves the action of death receptors and caspase-8. For example,alterations in healthy levels of proteins such as p53, Bim, Bax, Apaf-1,FLIP and many others have been observed in cancers. These alterationscan lead to a defective apoptotic cascade, one in which the upstreamproapoptotic signal is not adequately transmitted to activate theexecutioner caspases, caspase-3 and caspase-7.

As most apoptotic pathways ultimately involve the activation ofprocaspase-3, upstream genetic abnormalities are effectively “breaks” inthe apoptotic circuitry, and as a result such cells proliferateatypically. Given the central role of apoptosis in cancer, efforts havebeen made to develop therapeutics that target specific proteins in theapoptotic cascade. However, because these therapeutics target early (orintermediate to high) positions on the apoptotic cascade, cancers withmutations in proteins downstream of those members can still be resistantto the possible beneficial effects of those compounds.

For therapeutic purposes, it would be advantageous to identify smallmolecules that directly activate a proapoptotic protein far downstreamin the apoptotic cascade. This approach could involve a relatively lowposition in the cascade, thus enabling the killing of even those cellsthat have mutations in their upstream apoptotic machinery. Moreover,such therapeutic strategies would have a higher likelihood of success ifthat proapoptotic protein were upregulated in cancer cells. Thus,identifying small molecules that target the downstream effector proteinof apoptosis, procaspase-3, would significantly aid current cancertherapy.

The conversion or activation of procaspase-3 to caspase-3 results ingeneration of the active “executioner” caspase form that subsequentlycatalyzes the hydrolysis of a multitude of protein substrates. Incertain cancers, the levels of procaspase-3 are elevated relative tonormal tissue. A study of primary isolates from 20 colon cancer patientsrevealed that on average, procaspase-3 was upregulated six-fold in suchisolates relative to adjacent non-cancerous tissue. In addition,procaspase-3 is upregulated in certain neuroblastomas, lymphomas, andliver cancers. Furthermore, a systematic evaluation of procaspase-3levels in the 60 cell-line panel used for cancer screening by theNational Cancer Institute Developmental Therapeutics Program wasperformed. The evaluation revealed that certain lung, melanoma, renal,and breast cancers show greatly enhanced levels of procaspase-3expression. Due to the role of active caspase-3 in achieving apoptosis,the relatively high levels of procaspase-3 in certain cancerous celltypes, and the intriguing safety catch-mediated suppression of itsautoactivation, small molecules that directly modify procaspase-3, couldhave great applicability in targeted cancer therapy.

Furthermore, combination therapy has become increasingly important forthe treatment of cancer patients. The goal of combination therapy is toachieve an additive or synergistic effect between chemotherapeutics,thereby facilitating shortened treatment times, decreased toxicity, andincreased patient survival. Drugs that act on a single biochemicalpathway are particularly strong candidates for synergy or potentiationas they may mimic “synthetic lethal” genetic combinations. Thus, thereis an urgent a need for more effective therapies for the treatment ofmany forms of cancer, and new synergistic combinations of anticancerdrugs would aid this pursuit. Accordingly, there exists a need toidentify new combinations of cytotoxic agents that are effective inkilling cancer cells, yet protect normal host tissues from the undesiredtoxicity of the cytotoxic agent.

SUMMARY

The invention provides compositions that include a combination of activeagents for therapeutic cancer treatment. The compositions include smallmolecule drugs capable of inducing cancer cell death. The combination ofdrugs can be applicable to a variety of cancer diseases and cancer celltypes such as melanoma, adrenal, brain, breast, colorectal, esophageal,gallbladder, leukemia, liver, lung, lymphoma, neuroblastoma, ovarian,pancreatic, renal, thyroid, Erdheim-Chester disease (ECD),Langerhans'-cell histiocytosis (LCH), and others known in the art. Insome embodiments, the compositions interact directly or indirectly withprogrammed cell death pathway members such as procaspase-3. In variousembodiments, the compositions are selective for a particular type ofcancer cells, and can have reduced neurotoxicity compared to othercompounds that interact with programmed cell death pathway members suchas procaspase-3.

The combination anticancer therapy described herein can include drugsthat target different biochemical pathways, or drugs that hit differenttargets in the same pathway, mimicking “synthetic lethal” geneticcombinations. The combination of the procaspase-3 activator PAC-1 andinhibitors of the BRAF kinase that has the V600E mutation showsconsiderable synergy toward inducing apoptotic death of cancer cells toa degree well exceeding the additive effect. The combination of PAC-1and these inhibitors of the BRAF gene/enzyme can effectively reducetumor burden in tumor models in which the compounds alone have minimalor no effect. Data indicate significant efficacy for the combination ofPAC-1 and these inhibitors of the BRAF enzyme for the treatment ofcancer and, more broadly, show that this synergistic combination canprovide significantly heightened therapeutic benefits.

Accordingly, the invention provides a composition comprising (a) thecompound PAC-1:

-   -   (b) a second active agent, which agent is an inhibitor of the        BRAF enzyme that has a mutation; and    -   (c) a pharmaceutically acceptable diluent, excipient, or        carrier.

The inhibitor of the BRAF enzyme that has a mutation can be, forexample, vemurafenib, dabrafenib, BMS-908662 (Bristol-Myers Squibb),encorafenib (LGX818) (Novartis), PLX3603 (R05212054) (Hofmann-LaRoche),RAF265 (Novartis), sorafenib, or a derivative or prodrug of one of theaforementioned actives. Particularly effective inhibitors of the BRAFenzyme are inhibitors of the BRAF enzyme that has the V600E or the V600Kmutation. Such inhibitors include vemurafenib and dabrafenib. In otherembodiments, the composition further includes a MEK inhibitor, such astrametinib. Alternatively, the second active agent (which agent is aninhibitor of the BRAF enzyme that has a mutation) can be replaced with aMEK inhibitor such as trametinib to provide a distinct two-agentcomposition. In various embodiments, these actives can be administeredto a patient concurrently or consecutively. A carrier for thecomposition can include water and/or optional components foradvantageously delivering the actives such as a buffer, a sugar, acellulose, a cyclodextrin, or various combinations thereof. In oneembodiment, the cyclodextrin is 2-hydroxypropyl-β-cyclodextrin.

The invention also provides a method of inhibiting the growth orproliferation of cancer cells. This method includes contacting cancercells with an effective amount of a composition of described herein,wherein the composition can include one or both of PAC-1 (i.e., thefirst active agent) and one or more second active agents (e.g., aninhibitor of the BRAF enzyme that has a mutation, and/or a MEKinhibitor). When the composition includes only one of PAC-1 and thesecond active agent, the method can include subsequently contacting thecancer cells with the other(s). The method can thus also includecontacting cancer cells with an effective amount of a MEK inhibitor,concurrently or sequentially with PAC-1 and the second active agent.Contacting the cancer cells with these actives (e.g., PAC-1 and thesecond and/or third active agent) effectively inhibits the growth orproliferation of the cancer cells.

The invention further provides a method of inducing apoptosis in acancer cell. The method can include comprising contacting the cancercell with an effective amount of PAC-1 and an effective amount of asecond and/or third active agent, wherein apoptosis is thereby inducedin the cancer cell. The contacting can be in vitro. Alternatively, thecontacting can be in vivo. In one embodiment, the cancer cell can becontacted with PAC-1 and the second active agent concurrently. Inanother embodiment, the cancer cell can be contacted with the secondactive agent prior to contacting the cancer cell with PAC-1. In yetanother embodiment, the cancer cell can be contacted with PAC-1 prior tocontacting the cancer cell with the second active agent. The thirdactive agent (e.g., a MEK inhibitor) can be administered to the cancercell before or after PAC-1, and before or after the second active agent.

The invention also provides a method of treating a cancer in a patientin need thereof. The method includes administering to a patient,concurrently or sequentially, a therapeutically effective amount of acompound of PAC-1, and a second active agent, which agent is aninhibitor of the BRAF enzyme that has a mutation, for example, the V600Emutation or the V600K mutation, wherein the cancer is thereby treated.In certain specific embodiments, the second active agent is vemurafenibor dabrafenib:

As discussed above, PAC-1 and the second active agent can beadministered concurrently. In another embodiment, PAC-1 and the secondactive agent are administered sequentially. When administeredsequentially, the second active agent can be administered before PAC-1,or the second active agent can be administered after PAC-1. Inadditional embodiments, a therapeutically effective amount of a MEKinhibitor can be administered to the patient. The MEK inhibitor can beadministered concurrently or sequentially with respect to PAC-1 and thesecond active agent. Thus, in various embodiments, the MEK inhibitor canbe administered prior to, concurrently with, or after either PAC-1 orthe second active agent.

The cancer (or cancer cells, as the case may be) contacted or treatedcan be, for example, melanoma, adrenal cancer, brain cancer, breastcancer, colorectal cancer, esophageal cancer, gallbladder cancer, livercancer, lung cancer, lymphoma, neuroblastoma, ovarian cancer, pancreaticcancer, renal cancer, thyroid cancer, Erdheim-Chester disease (ECD),Langerhans'-cell histiocytosis (LCH), or leukemia, including hairy-cellleukemia. The melanoma can be a BRAFi-resistant melanoma, includingvemurafenib-resistant melanomas. The thyroid cancer can be papillarythyroid cancer. The lung cancer can be non-small cell lung cancer(NSCLC). In some embodiments, the cancer can be brain cancer, lymphoma,or cancer cells in bone tissue. For example, the cancer can beglioblastoma or oligodendroglioma. In another embodiment, cancer cellscan be osteosarcoma cells and the cancer treated is bone cancer. Othertypes of cancer cells that can be killed or inhibited, and othercancerous conditions that can be treated are described below.

The invention thus provides for the use of the compositions describedherein for use in medical therapy. The medical therapy can be treatingcancer, for example, melanoma and/or other cancers recited herein. Theinvention also provides for the use of a composition as described hereinfor the manufacture of a medicament to treat a disease in a mammal, forexample, cancer in a human. The medicament can include apharmaceutically acceptable diluent, excipient, or carrier.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the specification and are includedto further demonstrate certain embodiments or various aspects of theinvention. In some instances, embodiments of the invention can be bestunderstood by referring to the accompanying drawings in combination withthe detailed description presented herein. The description andaccompanying drawings may highlight a certain specific example, or acertain aspect of the invention. However, one skilled in the art willunderstand that portions of the example or aspect may be used incombination with other examples or aspects of the invention.

FIGS. 1A-C. The effect of vemurafenib and PAC-1 in ^(V600E)BRAF or^(WT)BRAF cell lines. (A) IC₅₀ values (5 day) of vemurafenib and PAC-1in a panel of nine cell lines. (B) and (C): Cell lines with ^(V600E)BRAFhave significantly higher percent of cells undergoing apoptosis(assessed by Annexin V-FITC/PI staining) after treatment withvemurafenib (10 μM) and PAC-1 (12 μM) (B), or vemurafenib (0.5 μM) andPAC-1 (12 μM) (C), for 24 h, whereas this combination has negligibleeffect on cell lines with wild-type BRAF. Dashed horizontal linesrepresent the level of cell death expected from a mere additive effectof the two agents. Values are reported as mean±SEM of at least threeindependent experiments. P-values shown for 2-way interaction todetermine if the combination for induction of apoptosis is differentfrom an additive effect (dashed horizontal lines) of individual agentsare statistically significant (* p<0.05, ** p<0.01, *** p<0.001).

FIGS. 2A-E. PAC-1+vemurafenib powerfully synergize to induce apoptoticdeath and caspase activity in A375 cells. (A) Shown is percent apoptoticcell death (assessed by Annexin V/PI staining and flow cytometry)induced after 24 h of treatment. Values shown are heat mapped with whiterepresenting low % apoptotic cell death and dark gray representing high% apoptotic cell death. (B) Combination indices (CI) calculated for eachcombination with Combosyn software. CI values are heat mapped withlowest values in light gray and the highest values in black. (C)Significant caspase-3/-7 enzymatic activity is observed in cells treatedwith the combination of PAC-1 and vemurafenib. PAC-1 (12 μM) andvemurafenib (10 μM) alone have little effect (p-values vs. DMSOcontrol >0.1 at all timepoints). Caspase-3/-7 activity in cell lysateswas assessed with the fluorogenic Ac-DEVD-AFC substrate. Activity isexpressed as normalized to minimal and maximal activity observed withinthe assay, with 1 μM staurosporine (STS) as the positive control.P-values shown for 2-way interaction to determine if the combination isdifferent from additive are statistically significant at indicatedtimepoints. (* p<0.05, ** p<0.01, *** p<0.001). (D) PAC-1 (12 μM) andvemurafenib (10 μM) alone have little effect on PARP-1 cleavage in A375cells, but significant PARP-1 cleavage is observed via Western blot withthe combination. (E) After 24 h, no/low inhibition of ERK1/2phosphorylation was observed at low concentrations of vemurafenib (0.1μM and 0.25 μM). At higher concentrations of vemurafenib (0.5 μM and 1μM), phosphorylation of ERK1/2 was effectively inhibited with or withoutaddition of PAC-1, indicating that effect of PAC-1 is downstream of theMAPK pathway. However, cleaved PARP-1 was only observed in cells treatedwith the vemurafenib/PAC-1 combination, even at concentrations ofvemurafenib (0.1 and 0.25 μM) where incomplete inhibition of ERK1/2phosphorylation was observed. Values are reported as mean±SEM of atleast three experiments.

FIGS. 3A-C. Addition of PAC-1 to the combination ofvemurafenib+trametinib powerfully synergizes to induce apoptotic deathand caspase activity in A375 and UACC-62 cells. (A) Shown is percentapoptotic cell death after 24 h of treatment. Combination of trametinib(100 nM) and vemurafenib (10 μM) leads to a minimal increase in thepopulation of apoptotic cells. Addition of PAC-1 (12 μM) leads to adramatic increase in the population of apoptotic cells that is beyondthe additive effect of the three agents. (B) Trametinib (100 nM) andvemurafenib (10 μM) in combination have little effect on PARP-1 cleavagein A375 and UACC-62 cells, but significant PARP-1 cleavage and reductionin procaspase-3 level are observed via Western blot with the addition ofPAC-1 (12 μM). (C) Combination of vemurafenib and trametinib lead toadditive increase in caspase-3/-7 activity but addition of PAC-1 leadsto significant increases in caspase-3/-7 enzymatic activity in A375 andUACC-62 after 24 h. PAC-1 (12 μM), vemurafenib (10 μM) and trametinib(100 nM) alone have little effect (p-values vs. DMSO control >0.1).Activity is expressed as normalized to the positive control. Dashedhorizontal lines represent the level of cell death expected from a mereadditive effect of the two agents. Values are reported as mean±SEM of atleast three experiments. P-values shown for 2-way interaction todetermine if the combination for induction of apoptosis is differentfrom an additive effect of individual agents are statisticallysignificant (* p<0.05, ** p<0.01, *** p<0.001).

FIGS. 4A-E. The PAC-1+vemurafenib combination retards tumor growth in anA375 subcutaneous mouse xenograft model of melanoma. (A) The effect ofPAC-1, vemurafenib, and their combination in the A375 model. Micebearing subcutaneous tumors were dosed for 15 days. Mice were dosed withPAC-1 once daily at 100 mg/kg (n=6) via i.p. injection, vemurafenibtwice daily at 10 mg/kg (n=8) by (p.o.), or the PAC-1+vemurafenibcombination (n=8). The black line below the x-axis indicates the dosingperiod for the mice during the study. Tumor volumes are plotted asmean±SEM. (B) Masses of the excised tumors. (C) Tumor lysates wereanalyzed by Western blot for changes in procaspase-3 levels. Actin wasused as loading control. Band intensity was quantified using ImageJ. (D)Plot of procaspase-3 levels normalized to the actin loading controls.(E) Percentage of cells that are positive for Ki-67 followingimmunohistochemical staining of formalin fixed tumor samples. 2000 cellswere counted in each sample for each of the four treatment groups.P-values shown are with respect to control mice. (* p<0.05, ** p<0.01,*** p<0.001).

FIGS. 5A-D. Low concentrations of PAC-1 (1 μM) significantly delay cellregrowth in combination with vemurafenib in long-term cell cultureexperiments. (A) Comparison of E_(max) values in A375 cells treated withvemurafenib and PAC-1. (B) A375 and SK-MEL-5 cells treated with PAC-1 (4μM) or vemurafenib (10 μM) for a duration of 30 days. (C) A375 cellswere treated with PAC-1 (1 vemurafenib (5 μM or 10 or the combination.After 5, 10 or 20 days, the wells were fixed with 10% trichloroaceticacid, stained with 0.5% sulforhodamine B (SRB) dye, and imaged withBioRad GelDoc RX. Day 20 images of control and PAC-1 samples are notshown because the cells were unviable due to overcrowding. (D)Quantification of (C) where the SRB dye is dissolved in 10 mM Tris baseat pH 10.4, and the absorbance read at 510 nm. Corrected absorbance at510 nm was plotted against the days of continuous treatment bynormalizing against absorbance on Day 0 before the start of treatment.Values are reported as mean±SEM of at least three experiments. T-testperformed between wells treated with vemurafenib only versus vemurafeniband PAC-1 (1 μM). On day 10, only the wells treated with vemurafenib (10μM) and PAC-1 (1 μM) are significantly different from vemurafenib (10μM) only (p=0.049) treatment. On day 20, wells treated with vemurafenib(5 or 10 μM) and PAC-1 (1 μM) are significantly different fromvemurafenib (5 or 10 μM), as indicated. (* p<0.05, ** p<0.01, ***p<0.001).

FIGS. 6A-D. PAC-1 retains activity in vemurafenib-resistant A375VR cells(A) Vemurafenib is significantly less active in A375R versus parentA375. (B) Treatment with 0.5 or 1 μM of vemurafenib is unable to inhibitphosphorylation of ERK1/2 in A375VR after 2 h. Under the sameconditions, complete inhibition of ERK1/2 phosphorylation was observedin the parental A375 cell line. (C) PAC-1 retains activity in the A375Rcell line. Values are reported as mean±SEM of at least three independentexperiments. (D) The effect of PAC-1, vemurafenib, and their combinationin the A375VR xenograft model. Mice bearing subcutaneous tumors weredosed for 15 days. Mice were dosed with PAC-1 twice daily at 100 mg/kg(n=7) by i.p. injection, vemurafenib twice daily at 10 mg/kg (n=5) by(p.o.), or the PAC-1+vemurafenib combination (n=5). The black line abovethe x-axis indicates the dosing period for the mice during the study.Tumor volumes are plotted as mean+SEM. P-values shown are with respectto control mice. (* p<0.05).

FIGS. 7A-E. PAC-1 and vemurafenib powerfully synergize to induceapoptotic death and caspase activity in SK-MEL-5 cells. (A) Shown ispercent apoptotic cell death (assessed by Annexin V/PI staining and flowcytometry) induced after 24 h of treatment. Values shown are heat mappedwith white representing low % apoptotic cell death and dark grayrepresenting high % apoptotic cell death. (B) Combination indices (CI)calculated for each combination with Combosyn software. CI values areheat mapped with lowest values in light gray and the highest values inblack. (C) Significant caspase-3/-7 enzymatic activity is observed incells treated with the combination of PAC-1 and vemurafenib; PAC-1 (12μM) and vemurafenib (10 μM) alone have little effect (p-values vs. DMSOcontrol >0.1 at all timepoints). Caspase-3/-7 activity in cell lysateswas assessed with the fluorogenic Ac-DEVD-AFC substrate. Activity isexpressed as normalized to minimal and maximal activity observed withinthe assay, with 1 μM staurosporine (STS) as the positive control. (D)PAC-1 (12 μM) and vemurafenib (10 μM) alone have little effect on PARP-1cleavage in SK-MEL-5 cells, but significant PARP-1 cleavage is observedvia western blot with the combination. (E) After 24 h, vemurafenib (0.5μM and 1 μM) inhibited the phosphorylation of ERK1/2 with or withoutaddition of PAC-1, indicating that effect of PAC-1 is downstream of theMAPK pathway. However, cleaved PARP-1 was only observed in cells treatedwith the vemurafenib/PAC-1 combination. Values are reported as mean±SEMof at least three independent experiments. P-values shown for 2-wayinteraction to determine if the combination is different from additiveare statistically significant at indicated timepoints. (* p<0.05, ***p<0.001).

FIGS. 8A-E. PAC-1 and vemurafenib powerfully synergize to induceapoptotic death and caspase activity in UACC-62 cells. (A) Shown ispercent apoptotic cell death (assessed by Annexin V/PI staining and flowcytometry) induced after 24 h of treatment. Values shown are heat mappedwith white representing low % apoptotic cell death and dark grayrepresenting high % apoptotic cell death. (B) Combination indices (CI)calculated for each combination with Combosyn software. CI values areheat mapped with lowest values in light gray and the highest values inblack. (C) Significant caspase-3/-7 enzymatic activity is observed incells treated with the combination of PAC-1 and vemurafenib PAC-1 (12μM) and vemurafenib (10 μM) alone have little effect (p-values vs. DMSOcontrol >0.1 at all timepoints). Caspase-3/-7 activity in cell lysateswas assessed with the fluorogenic Ac-DEVD-AFC substrate. Activity isexpressed as normalized to minimal and maximal activity observed withinthe assay, with 1 μM STS as the positive control. (D) PAC-1 (12 μM) andvemurafenib (10 μM) alone have little effect on PARP-1 cleavage inUACC-62 cells, but significant PARP-1 cleavage is observed via westernblot with the combination. (E) After 24 h, vemurafenib (0.5 μM and 1 μM)inhibited the phosphorylation of ERK1/2 with or without addition ofPAC-1, indicating that effect of PAC-1 is downstream of the MAPKpathway. Minimal cleaved PARP-1 was observed in PAC-1 only treatedcells, which was markedly increased in cells treated with thevemurafenib/PAC-1 combination. Values are reported as mean±SEM of atleast three independent experiments. P-values shown for 2-wayinteraction to determine if the combination is different from additiveare statistically significant at indicated timepoints. (*** p<0.001).

FIGS. 9A-D. Effect of PAC-1a (12 μM) vs PAC-1 (12 μM) in combinationwith vemurafenib (30 μM) in cell lines after 24 h treatment in (A) A375,(B) SK-MEL-5 and (C) UACC-62 cell lines as assessed by Annexin V-FITC/PIplots. Percent apoptosis reported is normalized relative to DMSO controlsample. Dashed horizontal lines represent the level of cell deathexpected from a mere additive effect of the two agents. (D) PAC-1 (12μM) and vemurafenib (10 μM) alone have minimal effect on PARP-1 cleavagein A375 cells, but increased PARP-1 cleavage is observed with thecombination. PAC-1a (12 μM) in combination with vemurafenib (10 μM) doesnot increase PARP-1 cleavage. Values are reported as mean±SEM of atleast three independent experiments. P-values shown for 2-wayinteraction to determine if the combination for induction of apoptosisis different from an additive effect (dashed horizontal lines) ofindividual agents are statistically significant (*** p<0.001).

FIGS. 10A-C. Effect of the PAC-1 and vemurafenib combination in MIAPaCa-2 (mutant KRAS and ^(WT)BRAF) and CHL-1 (^(WT)KRAS and ^(WT)BRAF)cell lines with ^(WT)BRAF. (A) No effect on procaspase-3 activation isobserved in MIA PaCa-2 and CHL-1 cell lines when treated with PAC-1 (12μM)+vemurafenib (30 Caspase-3/-7 activity in cell lysates was assessedwith the fluorogenic Ac-DEVD-AFC substrate. Activity is expressed asnormalized to minimal and maximal activity observed within the assay,with 1 μM STS as the positive control. (B) No effect on PARP-1 cleavagewas observed in MIA PaCa-2 cells after 24 h. (C) PAC-1 (12 μM) andvemurafenib (30 μM) have no effect on PARP-1 cleavage in CHL-1 cellsafter 24 h treatment. Values are reported as mean±SEM of at least threeindependent experiments.

FIG. 11. Images of tumor-bearing mice that were sacrificed after 15 daysof continuous dosing. The four treatment groups are: control (n=6, 0mg/kg PAC-1 and vemurafenib); mice treated once-a-day with 100 mg/kgPAC-1 (n=6), twice-a-day with 10 mg/kg vemurafenib (n=8), and thecombination of 100 mg/kg PAC-1 (once-a-day) and 10 mg/kg vemurafenib(twice-a-day) (n=8).

FIGS. 12A-B. Addition of PAC-1 (1 μM) in the long-term treatment ofUACC-62 cells with vemurafenib significantly delays cell regrowth. (A)UACC-62 cells were treated with PAC-1 (1 μM), vemurafenib (5 μM or 10μM), or the combination. Media was washed out every 2-3 days and newcompounds were added into each well. After 5, 10 or 20 days, the wellswere fixed with 10% trichloroacetic acid, stained with 0.5%sulforhodamine B (SRB) dye, and imaged with BioRad GelDoc RX. Day 20images of control and PAC-1 samples are not shown because the cells wereunviable due to overcrowding. (B) Quantification of (A) where the SRBdye is dissolved in 10 mM Tris base at pH 10.4, and the absorbance readat 510 nm. Corrected absorbance at 510 nm was plotted against the daysof continuous treatment by normalizing against absorbance on Day 0before the start of treatment. Values are reported as mean±SEM of atleast three independent experiments. 2-tailed t-test performed betweenwells treated with vemurafenib only versus vemurafenib and PAC-1 (1 μM).On day 10, only the wells treated with vemurafenib (10 μM) and PAC-1 (1μM) is significantly different from vemurafenib (10 μM) only (p=0.035)treatment. On day 20, wells treated with vemurafenib (5 or 10 μM) andPAC-1 (1 μM) are significantly different from vemurafenib (5 or 10 μM),as indicated on the graph. (* p<0.05, *** p<0.001).

FIGS. 13A-C. Effect of the PAC-1 and vemurafenib combination in A375VRcells. (A) Shown is percent apoptotic cell death (assessed by AnnexinV/PI staining and flow cytometry) induced after 24 h of treatment. (B)The apoptotic cell death observed in (A) is greater than that predictedby the Bliss independent model. The excess cell death is calculated as[f_((observed, apoptotic))−(f_((PAC-1, apoptotic))+f_((vemurafenib, apoptotic))−f_((PAC-1, apoptotic))*f_((vemurafenib, apoptotic)))]*100%.This indicates that the observed effect is synergistic rather thanadditive. (C) The synergistic effect of PAC-1 and vemurafenib inactivating apoptosis in A375VR after 24 h. This effect is abolished whenthe inactive PAC-1a was used. Dashed horizontal lines represent thelevel of cell death expected from a mere additive effect of the twoagents. Values are reported as mean±SEM of at least three independentexperiments. P-values shown for 2-way interaction to determine if thecombination for induction of apoptosis is different from an additiveeffect (dashed horizontal lines) of individual agents are statisticallysignificant (* p<0.05, ** p<0.01, *** p<0.001).

DETAILED DESCRIPTION

Many cancers resist standard chemotherapy, or become resistant to aparticular chemotherapeutic after a period of time. The combinationtherapy described herein takes advantage of the procaspase-1 activationby PAC-1, which can synergize with the chemotherapeutic properties of asecond active agent such as an inhibitor of the BRAF enzyme that has amutation, to provide efficacy under conditions where one of the activesalone might be less effective or completely ineffective. These compoundscan also be successful in targeted cancer therapy, where there can beadvantages of selectivity in the killing of cancer cells with comparablyreduced adverse reactions to non-cancerous cells having lower levels ofprocaspase-3. These reduced adverse reactions can include reductions intoxicity, particularly neurotoxicity.

The combination of compounds, the compositions and the methods describedherein can act via modulation of apoptosis or programmed cell death andother chemotherapeutic mechanisms to be effective in the treatment ofcancer. In one embodiment, the modulation of apoptosis is by inductionor activation of apoptosis. In various embodiments, the administrationof compounds can be concurrent, or alternatively, sequential.

The invention thus provides methods for potentiation of an active agentby PAC-1, for example, for the treatment of melanoma, colorectal cancer,thyroid cancer, lung cancer, or ovarian cancer. During apoptosis, thezymogen procaspase-3 is activated via proteolysis to caspase-3, and thisactive caspase-3 then cleaves scores of cellular substrates, executingthe apoptotic program. Because procaspase-3 protein levels are elevatedin various tumor histologies, drug-mediated direct activation ofprocaspase-3 can be highly effective as a selective anticancer strategy.

Certain compounds can enhance the activity and automaturation ofprocaspase-3 and induce apoptosis in cancer cells. Procaspase-activatingcompound-1 (PAC-1) enhances the activity of procaspase-3 via thechelation of inhibitory zinc ions, induces apoptosis in cancer cells inculture, and has efficacy in multiple murine tumor models. Novelcombinations of PAC-1 and inhibitors of the BRAF enzyme that has amutation have been found to be synergistically effective in treatingcancer cells, as described herein. Because PAC-1 acts late in theapoptotic cascade, it is uniquely capable of synergizing with a widerange of chemotherapeutic active agents, as described herein.

Melanoma is the most common cutaneous malignancy and upon metastasis isconsidered the deadliest form of skin cancer. It is the fifth mostcommon cancer in the United States. One common mutation in melanoma isthe substitution of a valine for glutamate (V600E) in the kinase domainof the BRAF protein (Davies et al., Nature 2002, 417, 949). The V600Emutation constitutively activates BRAF and the downstream MEK-ERKsignaling pathway, leading to tumorigenesis. The discovery thatapproximately 50% of melanomas harbor the V600E mutation in the BRAFprotein spurred the development of ^(V600E)BRAF inhibitors, and thesubsequent approval of vemurafenib in 2011. ^(V600E)BRAF inhibitors likevemurafenib (and dabrafenib, approved in 2013) lead to impressivereduction in tumor burden within weeks of therapy, and extension ofprogression-free survival by three to four months.

Despite their initial anti-melanoma activity, resistance to ^(V600E)BRAFinhibitors rapidly emerges. In the majority of resistant tumors,reactivation of the MAPK signaling pathway is observed, motivating theaddition of MEK1/2 inhibitors (e.g., trametinib) to the treatmentregimen for metastatic melanoma. Upfront combination therapy with MEK1/2and ^(6V00E)BRAF inhibitors is effective in delaying the median time toresistance by 3.7 to 4.1 months in patients who have not received prior^(V600E)BRAF inhibition treatment, but the addition of MEK1/2 inhibitorto patients who have already failed prior ^(V600E)BRAF inhibitor therapyonly results in a marginal improvement in anticancer efficacy. Given thecurrent clinical limitations of existing therapies, novel andrationally-designed combination studies with other kinase inhibitors arebeing explored. Despite all efforts to date, the development ofresistance to targeted ^(V600E)BRAF therapies emerges in virtually 100%of patients treated; acquired drug resistance to this class of agentsremains a significant obstacle to dramatically enhanced survivalbenefits for metastatic melanoma patients.

In contrast to many studies that have focused on the combination ofvemurafenib with inhibitors of diverse and druggable kinases,combination therapy of vemurafenib with agents that activate theapoptotic pathway have not been extensively explored. In part, this lackof exploration might be attributed to the fact that melanoma cellspossess multiple defects in their apoptotic signaling pathways,rendering them resistant to many proapoptotic stimuli. We hypothesizedthat a suitable proapoptotic agent that induces apoptosis downstream ofthese apoptotic defects would be highly synergistic with ^(6V00E)BRAFinhibitors.

Given that the aberrations in the apoptotic signaling cascades inmelanoma cells are upstream of the activation of procaspase-3, drugsthat directly activate procaspase-3 are intriguing candidates for thiscombination therapy. In addition, because melanomas have elevatedexpression of procaspase-3, a procaspase-3 activator should be potentand selective for such cells. Furthermore, it is known that ^(V600E)BRAFinhibitors induce apoptotic cell death mediated by caspase-3; thus, thecombination of vemurafenib with a direct procaspase-3 activator couldlead to dramatically enhanced caspase-3 activity and cancer cell deathrelative to the effect of either single-agent. PAC-1 is a small moleculethat directly activates cellular procaspase-3 via chelation of labileinhibitory zinc. Due to the overexpression of procaspase-3 in cancers ofdiverse origins, PAC-1 and its derivatives selectively induce apoptosisin cancer cells while sparing non-cancerous cells. PAC-1 exerts singleagent activity in multiple murine models of cancer, including axenograft model of melanoma. Importantly, in addition to favorablepreclinical activity in murine tumor models, human cancer patients havebeen taking PAC-1 as part of a Phase I clinical trial since March 2015(NCT02355535).

Vemurafenib, the first approved BRAF inhibitor, is a targeted therapyfor melanoma patients who have the V600E BRAF protein (Bollag et al.,Nat. Rev. Drug Discov. 2012, 11, 873). Treatment with vemurafenib leadsto apoptosis and rapid tumor regression, extending the progression-freesurvival of melanoma patients with the V600E BRAF protein by 5.3 months(McArthur et al., Lancet Oncol. 2014, 15, 323). While vemurafenibrepresents a significant advance in the treatment of melanoma, onset ofresistance has been a significant concern in the clinic. Combinationtherapy of vemurafenib with an MEK inhibitor has been clinically testedto extend the duration of progression-free survival by 3.7 months, butresistance ultimately arises due to reactivation of the RAF-MEK-ERKpathway (Larkin et al., N. Engl. J. Med. 2014, 371, 1867).

Elevated expression of procaspase-3, the executioner caspase in theapoptotic cascade, has been reported in various cancers includingmelanoma (Fink et al., Melanoma Res. 2001, 11, 385; Chen et al., Hum.Pathol. 2009, 40, 950). Small molecule activation of procaspase-3 istherefore an attractive therapeutic strategy for melanoma due to the keyrole played by procaspase-3 in the apoptotic cascade. Procaspase-3activating compound 1 (PAC-1) is a small molecule that chelates thelabile pool of zinc ions, which inhibit procapase-3, thus priming cancercells for apoptotic death (Peterson et al., J. Mol. Biol. 2009, 388,144). PAC-1 has shown single agent efficacy in a murine xenograft modelof melanoma, validating the potential of procaspase-3 activation as ananti-cancer strategy (Wang et al., Mol. Oncol. 2014, 8, 1640). Giventhat PAC-1 primes cells for apoptotic death and vemurafenib inducesapoptosis in cancer cells, we find that vemurafenib in combination withPAC-1 dramatically enhances therapeutic efficacy.

We recently discovered that PAC-1 shows outstanding synergy withinhibitors of the BRAF enzyme that has the V600E mutation, includingvemurafenib (marketed as zelboraf), a drug that was recently approvedfor the treatment of melanoma. Based on our data (see FIGS. 1-13), PAC-1will show equivalent synergy with all drugs in this class (inhibitors ofthe BRAF enzyme that has the V600E or the V600K mutation), which alsoincludes dabrafenib (trade name tafinlar) and others.

The synergistic activity of inhibitors of the BRAF enzyme that has theV600E mutation, such as vemurafenib, with PAC-1 in enhancing apoptoticcell death in a variety of melanoma cell lines containing the V600E BRAFprotein is described herein. Importantly, PAC-1 retains activity invemurafenib-resistant A375R cell line, indicating its utility inmelanomas that have progressed beyond BRAF-inhibitor treatment.

Definitions

The following definitions are included to provide a clear and consistentunderstanding of the specification and claims. As used herein, therecited terms have the following meanings. All other terms and phrasesused in this specification have their ordinary meanings as one of skillin the art would understand. Such ordinary meanings may be obtained byreference to technical dictionaries, such as Hawley's Condensed ChemicalDictionary 14^(th) Edition, by R. J. Lewis, John Wiley & Sons, New York,N.Y., 2001.

References in the specification to “one embodiment”, “an embodiment”,etc., indicate that the embodiment described may include a particularaspect, feature, structure, moiety, or characteristic, but not everyembodiment necessarily includes that aspect, feature, structure, moiety,or characteristic. Moreover, such phrases may, but do not necessarily,refer to the same embodiment referred to in other portions of thespecification. Further, when a particular aspect, feature, structure,moiety, or characteristic is described in connection with an embodiment,it is within the knowledge of one skilled in the art to affect orconnect such aspect, feature, structure, moiety, or characteristic withother embodiments, whether or not explicitly described.

The singular forms “a,” “an,” and “the” include plural reference unlessthe context clearly dictates otherwise. Thus, for example, a referenceto “a compound” includes a plurality of such compounds, so that acompound X includes a plurality of compounds X. It is further noted thatthe claims may be drafted to exclude any optional element. As such, thisstatement is intended to serve as antecedent basis for the use ofexclusive terminology, such as “solely,” “only,” and the like, inconnection with any element described herein, and/or the recitation ofclaim elements or use of “negative” limitations.

The term “and/or” means any one of the items, any combination of theitems, or all of the items with which this term is associated. Thephrases “one or more” and “at least one” are readily understood by oneof skill in the art, particularly when read in context of its usage. Forexample, the phrase can mean one, two, three, four, five, six, ten, 100,or any upper limit approximately 10, 100, or 1000 times higher than arecited lower limit. For example, one or more substituents on a phenylring refers to one to five, or one to four, for example if the phenylring is disubstituted.

The term “about” can refer to a variation of ±5%, ±10%, ±20%, or ±25% ofthe value specified. For example, “about 50” percent can in someembodiments carry a variation from 45 to 55 percent. For integer ranges,the term “about” can include one or two integers greater than and/orless than a recited integer at each end of the range. Unless indicatedotherwise herein, the term “about” is intended to include values, e.g.,weight percentages, proximate to the recited range that are equivalentin terms of the functionality of the individual ingredient, thecomposition, or the embodiment. The term about can also modify theend-points of a recited range as discuss above in this paragraph.

As will be understood by the skilled artisan, all numbers, includingthose expressing quantities of ingredients, properties such as molecularweight, reaction conditions, and so forth, are approximations and areunderstood as being optionally modified in all instances by the term“about.” These values can vary depending upon the desired propertiessought to be obtained by those skilled in the art utilizing theteachings of the descriptions herein. It is also understood that suchvalues inherently contain variability necessarily resulting from thestandard deviations found in their respective testing measurements.

As will be understood by one skilled in the art, for any and allpurposes, particularly in terms of providing a written description, allranges recited herein also encompass any and all possible sub-ranges andcombinations of sub-ranges thereof, as well as the individual valuesmaking up the range, particularly integer values. A recited range (e.g.,weight percentages or carbon groups) includes each specific value,integer, decimal, or identity within the range. Any listed range can beeasily recognized as sufficiently describing and enabling the same rangebeing broken down into at least equal halves, thirds, quarters, fifths,or tenths. As a non-limiting example, each range discussed herein can bereadily broken down into a lower third, middle third and upper third,etc. As will also be understood by one skilled in the art, all languagesuch as “up to”, “at least”, “greater than”, “less than”, “more than”,“or more”, and the like, include the number recited and such terms referto ranges that can be subsequently broken down into sub-ranges asdiscussed above. In the same manner, all ratios recited herein alsoinclude all sub-ratios falling within the broader ratio. Accordingly,specific values recited for radicals, substituents, and ranges, are forillustration only; they do not exclude other defined values or othervalues within defined ranges for radicals and substituents.

One skilled in the art will also readily recognize that where membersare grouped together in a common manner, such as in a Markush group, theinvention encompasses not only the entire group listed as a whole, buteach member of the group individually and all possible subgroups of themain group. Additionally, for all purposes, the invention encompassesnot only the main group, but also the main group absent one or more ofthe group members. The invention therefore envisages the explicitexclusion of any one or more of members of a recited group. Accordingly,provisos may apply to any of the disclosed categories or embodimentswhereby any one or more of the recited elements, species, orembodiments, may be excluded from such categories or embodiments, forexample, for use in an explicit negative limitation.

The term “contacting” refers to the act of touching, making contact, orof bringing to immediate or close proximity, including at the cellularor molecular level, for example, to bring about a physiologicalreaction, a chemical reaction, or a physical change, e.g., in asolution, in a reaction mixture, in vitro, or in vivo.

“Concurrently” means (1) simultaneously in time, or (2) at differenttimes during the course of a common treatment schedule.

“Sequentially” refers to the administration of one active agent used inthe method followed by administration of another active agent. Afteradministration of one active agent, the next active agent can beadministered substantially immediately after the first, or the nextactive agent can be administered after an effective time period afterthe first active agent; the effective time period is the amount of timegiven for realization of maximum benefit from the administration of thefirst active agent.

An “effective amount” refers to an amount effective to treat a disease,disorder, and/or condition, or to bring about a recited effect, such asactivation or inhibition. For example, an effective amount can be anamount effective to reduce the progression or severity of the conditionor symptoms being treated. Determination of a therapeutically effectiveamount is well within the capacity of persons skilled in the art. Theterm “effective amount” is intended to include an amount of a compounddescribed herein, or an amount of a combination of compounds describedherein, e.g., that is effective to treat a disease or disorder, or totreat the symptoms of the disease or disorder, in a host. Thus, an“effective amount” generally means an amount that provides the desiredeffect.

In one embodiment, an effective amount refers to an amount of the activeagent described herein that are effective, either alone or incombination with a pharmaceutical carrier, upon single- or multiple-doseadministration to a cell or a subject, e.g., a patient, at inhibitingthe growth or proliferation, inducing the killing, or halting the growthof hyperproliferative cells. Such growth inhibition or killing can bereflected as a prolongation of the survival of the subject, e.g., apatient beyond that expected in the absence of such treatment, or anyimprovement in the prognosis of the subject relative to the absence ofsuch treatment.

The terms “treating”, “treat” and “treatment” include (i) inhibiting thedisease, pathologic or medical condition or arresting its development;(ii) relieving the disease, pathologic or medical condition; and/or(iii) diminishing symptoms associated with the disease, pathologic ormedical condition. Thus, the terms “treat”, “treatment”, and “treating”can include lowering, stopping or reversing the progression or severityof the condition or symptoms being treated. As such, the term“treatment” can include medical and/or therapeutic administration, asappropriate. In some embodiments, the terms “treatment”, “treat” or“treated” can refer to (i) a reduction or elimination of symptoms or thedisease of interest (therapy) or (ii) the elimination or destruction ofthe tumor (cure).

The terms “inhibit”, “inhibiting”, and “inhibition” refer to theslowing, halting, or reversing the growth or progression of a disease,infection, condition, or group of cells. The inhibition can be greaterthan about 20%, 40%, 60%, 80%, 90%, 95%, or 99%, for example, comparedto the growth or progression that occurs in the absence of the treatmentor contacting. Additionally, the terms “induce,” “inhibit,”“potentiate,” “elevate,” “increase,” “decrease,” or the like denotequantitative differences between two states, and can refer to at leaststatistically significant differences between the two states. Forexample, “an amount effective to inhibit the growth ofhyperproliferative cells” means that the rate of growth of the cells canbe, in some embodiments, at least statistically significantly differentfrom the untreated cells. Such terms can be applied herein to, forexample, rates of proliferation.

The phrase “inhibiting the growth or proliferation” of thehyperproliferative cell, e.g. neoplastic cell, refers to the slowing,interrupting, arresting, or stopping its growth and metastasis, and doesnot necessarily indicate a total elimination of the neoplastic growth.

The term “cancer” generally refers to any of a group of more than 100diseases caused by the uncontrolled growth of abnormal cells. Cancer cantake the form of solid tumors and lymphomas, and non-solid cancers suchas leukemia. Unlike normal cells, which reproduce until maturation andthen only as necessary to replace wounded cells, cancer cells can growand divide endlessly, crowding out nearby cells and eventually spreadingto other parts of the body.

The invention provides methods for treating cancer and cancerousconditions, and particularly cancers that carry the V600E BRAF proteinor the V600K BRAF protein. The term “cancerous condition” relates to anycondition where cells are in an abnormal state or condition that ischaracterized by rapid proliferation or neoplasia. A cancerous conditionmay be malignant or non-malignant (e.g. precancerous condition) innature. To farther describe a “cancerous condition”, the terms“hyperproliferative”, “hyperplastic”, “hyperplasia”, “malignant”,“neoplastic” and “neoplasia” can be used. These terms can be usedinterchangeably and are meant to include all types of hyperproliferativegrowth, hyperplastic growth, cancerous growths or oncogenic processes,metastatic tissues or malignantly transformed cells, tissues or organs,irrespective of histopathologic type, stage of invasiveness, orcancerous determination (e.g. malignant and nonmalignant).

The term “neoplasia” refers to new cell growth that results in a loss ofresponsiveness to normal growth controls, e.g., neoplastic cell growth.A “hyperplasia” refers to cells undergoing an abnormally high rate ofgrowth. However, these terms can be used interchangeably, as theircontext will reveal, referring generally to cells experiencing abnormalcell growth rates. “Neoplasias” and “hyperplasias” include tumors, whichmay be either benign, premalignant, carcinoma in-situ, malignant, solidor non-solid. Examples of some cancerous conditions that are can betreated include, but are not limited to, anal cancer, transitional cellbladder cancer, bone cancer, breast cancer, cervical cancer, colorectalcancer, gastric cancer, head and neck cancer, Kaposi's sarcoma,leukemia, lung cancer such as bronchogenic lung cancer, small cell lungcancer, and non-small cell lung cancer, Hodgkin's lymphoma,Non-Hodgkin's lymphoma, malignant lymphoma, neuroblastomas, osteogeniccarcinomas (e.g. cancer of the bone), ophthalmic cancers (e.g.retinoblastomas and other cancers of the eye), ovarian cancer, prostatecancer, renal cancer, skin cancers such as melanoma, soft tissuesarcomas, thyroid cancer, and Wilms' tumor. Other examples ofnon-malignant hyperproliferative conditions (e.g. precancerousconditions) that are within the scope of the invention include, but arenot limited to, adenomas, chondromas, enchondromas, fibromas, myomas,myxomas, neurinomas, osteoblastomas, osteochondromas, osteomas,papillary tumors, and the like, including other cancers describedherein.

The terms “leukemia” or “leukemic cancer” refer to all cancers orneoplasias of the hematopoetic and immune systems (blood and lymphaticsystem). These terms refer to a progressive, malignant disease of theblood-forming organs, marked by distorted proliferation and developmentof leukocytes and their precursors in the blood and bone marrow.Myelomas refer to other types of tumors of the blood and bone marrowcells. Lymphomas refer to tumors of the lymph tissue. Examples ofleukemia include acute myelogenous leukemia (AML), acute lymphoblasticleukemia (ALL), and chronic myelogenous leukemia (CIVIL).

As described herein, the compositions and methods of the invention canbe used for the treatment of various neoplasia disorders including suchconditions as acral lentiginous melanoma, actinic keratoses,adenocarcinoma, adenoid cycstic carcinoma, adenomas, adenosarcoma,adenosquamous carcinoma, astrocytic tumors, bartholin gland carcinoma,basal cell carcinoma, bronchial gland carcinomas, capillary, carcinoids,carcinoma, carcinosarcoma, cavernous, cholangiocarcinoma, chondosarcoma,choriod plexus papilloma/carcinoma, clear cell carcinoma, cystadenoma,endodermal sinus tumor, endometrial hyperplasia, endometrial stromalsarcoma, endometrioid adenocarcinoma, ependymal, epitheloid, Ewing'ssarcoma, fibrolamellar, focal nodular hyperplasia, gastrinoma, germ celltumors, glioblastoma, glucagonoma, hemangiblastomas,hemangioendothelioma, hemangiomas, hepatic adenoma, hepaticadenomatosis, hepatocellular carcinoma, insulinoma, intaepithelialneoplasia, interepithelial squamous cell neoplasia, invasive squamouscell carcinoma, large cell carcinoma, leiomyosarcoma, lentigo malignamelanomas, malignant melanoma, malignant mesothelial tumors,medulloblastoma, medulloepithelioma, melanoma, meningeal, mesothelial,metastatic carcinoma, mucoepidermoid carcinoma, neuroblastoma,neuroepithelial adenocarcinoma nodular melanoma, oat cell carcinoma,oligodendroglial, osteosarcoma, pancreatic polypeptide, papillary serousadenocarcinoma, pineal cell, pituitary tumors, plasmacytoma,pseudosarcoma, pulmonary blastoma, renal cell carcinoma, retinoblastoma,rhabdomyosarcoma, sarcoma, serous carcinoma, small cell carcinoma, softtissue carcinomas, somatostatin-secreting tumor, squamous carcinoma,squamous cell carcinoma, submesothelial, superficial spreading melanoma,undifferentiated carcinoma, uveal melanoma, verrucous carcinoma, vipoma,well differentiated carcinoma, and Wilm's tumor. Accordingly, thecompositions and methods described herein can be used to treat skincancer, bladder cancer, brain cancer (including intracranial neoplasmssuch as glioma, meninigioma, neurinoma, and adenoma), breast cancer,colon cancer, lung cancer (SCLC or NSCLC), ovarian cancer, pancreaticcancer, prostate cancer, and/or other cancers recited herein.

In some embodiments, the combination of PAC-1 and a second active agent(e.g., an inhibitor of the BRAF enzyme that has a mutation, for example,the active agent vemurafenib) can be particularly effective for treatingmelanoma. Other cancers that can be treated include, but are not limitedto, oligodendrogliomas and glioblastomas including glioblastomamultiforme (GBM). Tissues affected by the cancerous cells can be in thebrain itself (e.g., the cranium or the central spinal canal) or inlymphatic tissue, in blood vessels, in the cranial nerves, in the brainenvelopes (meninges), skull, pituitary gland, or pineal gland. Specificforms of brain cancer that can be treated include astrocytomas,chondromas, chondrosarcomas, chordomas, CNS (central nervous system)lymphomas, craniopharyngiomas, ependymomas, gangliogliomas,ganglioneuromas (also called gangliocytomas), gliomas, includingastrocytomas, oligodendrogliomas, and ependymomas, hemangioblastomas(also called vascular tumors), primitive neuroectodermal tumors (PNET)such as medulloblastomas, meningiomas, and vestibular schwannomas(formerly known as acoustic neuroma/schwannoma).

The combination can also be used to treat metastatic tumors that invadethe intracranial sphere from cancers originating in other organs of thebody. These conditions are typically referred to as secondary braintumors. Secondary brain tumors that can be treated with the combinationof PAC-1 and a second active agent include metastatic tumors of thebrain that originate from lung cancer, breast cancer, malignantmelanoma, kidney cancer, colon cancer, and other carcinomas.

Other examples of cancerous conditions that are within the scope of theinvention include, but are not limited to, neuroblastomas and osteogeniccarcinomas (e.g. cancer of the bone or neoplastic growth of tissue inbone). Examples of malignant primary bone tumors that can be treatedwith the combination of PAC-1 and a second active agent includeosteosarcomas, chondrosarcomas, Ewing's sarcoma, fibrosarcomas, and thelike, and secondary bone tumors such as metastatic lesions that havespread from other organs, including carcinomas of the breast, lung, andprostate.

Therapeutic Agents and Activity

Procaspase-activating compound-1 (PAC-1;(2-(4-benzylpiperazin-1-yl)-N-[(2-hydroxy-3-prop-2-enyl-phenyl)methylideneamino]acetamide)selectively induces apoptosis in cancerous cells. Methods of preparingPAC-1 are described in U.S. Pat. No. 8,778,945 (Hergenrother et al.).PAC-1 enhances the activity of procaspase-3 via the chelation ofinhibitory zinc ions, induces apoptosis in cancer cells. PAC-1 canenhance the activity and automaturation of procaspase-3 and induceapoptosis in cancer cells. PAC-1 also enhances the chemotherapeuticactivity of inhibitors of the BRAF enzyme having a mutation (the secondactive), often where either PAC-1 or the second active is less effectiveor completely inactive alone.

It was surprisingly discovered that PAC-1 and its derivatives cansynergize the activity of inhibitors of the BRAF enzyme having amutation. Accordingly, the invention provides further embodiments wherethe active agent PAC-1 in the compositions described herein can beexchanged for a PAC-1 derivative as described in U.S. Pat. No. 8,592,584(Hergenrother et al.) or U.S. Pat. No. 8,778,945 (Hergenrother et al.),which patents are incorporated herein by reference, to provideadditional compositions of the invention. One example of such PAC-1derivatives is SPAC-1(4-((4-(2-(2-(3-allyl-2-hydroxybenzylidene)hydrazinyl)-2-oxoethyl)piperazin-1-yl)methyl)benzenesulfonamide).PAC-1 and its derivatives can also synergize the activity of MEKinhibitors such as trametinib.

Accordingly, PAC-1 can be combined with an inhibitor of the BRAF enzymethat has a mutation, as described herein, and/or with an MEK inhibitorsuch as trametinib, cobimetinib, binimetinib (MEK162), selumetinib,PD-325901, CI-1040, PD035901, or TAK-733. MEK inhibitors are drugs thatinhibit the mitogen-activated protein kinase kinase enzymes MEK1 and/orMEK2. They can be used to affect the MAPK/ERK pathway, which isoveractive in certain cancers.

The amount or concentration of PAC-1 or the PAC-1 derivative in atherapeutic composition can be the amount or concentration effective toinhibit cancer cell growth, to induce apoptosis in a cancer cell, or tosynergize with the second active agent. For example, the concentrationof PAC-1 can be about 0.2 μM to about 5 mM, or about 2 μM to about 50μM, typically about 2.5 μM, about 5 μM, about 7.5 μM, about 10 μM, about12.5 μM, about 15 μM, about 20 μM, about 25 μM, about 30 μM, about 40μM, or about 50 μM, or a range between any of the aforementioned values.Similarly, the concentration of the second active agent (e.g.,inhibitors of the BRAF enzyme having a mutation such as vemurafenib ordabrafenib) can be about 1 nM to about 1 mM, or about 25 nM to about 1mM, typically about 1 nM, about 2 nM, about 3 nM, about 5 nM, about 10nM, about 25 nM, about 50 nM, about 100 nM, about 250 nM, about 500 nM,about 750 nM, about 900 nM, about 1 μM, about 2.5 μM, about 5 μM, about7.5 μM, about 10 μM, about 12.5 μM, about 15 μM, about 20 μM, about 25μM, about 30 μM, about 40 μM, about 50 μM, about 75 μM, about 100 μM,about 125 μM, about 150 μM, about 200 μM, about 250 μM, about 300 μM,about 500 μM, about 750 μM, or about 1 mM, or a range between any of theaforementioned values. One of skill in the art can readily convert theamount of active agent in a dose of a particular concentration to anamount of active agent, for example, for use in a solid dosage unit.

Data for various experiments are shown in FIGS. 1-13. PAC-1 andvemurafenib powerfully synergize to induce apoptotic death and caspaseactivity in melanoma cells. A dramatic procaspase-3 activation isobserved in cells treated with PAC-1+vemurafenib. Additionally, 12 μMPAC-1 and 10 μM vemurafenib alone have little effect on PARP-1 cleavagein A375 cells, but significant PARP cleavage is observed (via westernblot) with the combination. Furthermore, the addition of PAC-1 to thecombination of vemurafenib and an MEK inhibitor, trametinib,significantly enhances the caspase-3 activity and proapoptotic effect ofthe combination. Moreover, addition of low concentrations of PAC-1delays the regrowth of cancer cells following treatment withvemurafenib. PAC-1 also remains potent against vemurafenib-resistantA375VR cells in cell culture and synergizes with vemurafenib to exertantitumor effects on A375VR cell growth in vivo. Our data indicate thatinhibition of MAPK signaling combined with concurrent procaspase-3activation is an effective strategy to enhance the antitumor activity ofvemurafenib and mitigate the development of resistance. Accordingly, theinvention provides a method of overcoming vemurafenib resistance byadministering PAC-1 in combination with vemurafenib therapy, and/orvemurafenib/MEK inhibitor therapy to patients having vemurafenibresistant cancer.

Methods of the Invention

The invention provides methods of selectively inducing apoptosis in acancer cell, comprising administering to a cancer cell a combination ofcompounds capable of modifying a procaspase-3 molecule of said cancercell; wherein the combination of compounds is PAC-1 and a second activeagent. Also provided is a method of selectively inducing apoptosis in acancer cell, comprising administering to a cancer cell a combination ofcompounds capable of modifying a procaspase-3 molecule of the cancercell; wherein the combination of compounds is PAC-1 and a second activeagent, for example, wherein the cancer cell is in a patient in need oftreatment.

The invention provides additional methods where the recited combinationof compounds is PAC-1 and a second active agent, for example, as amethod of treating a cancer cell, comprising (a) identifying a potentialsusceptibility to treatment of a cancer cell with a procaspase activatorcompound; and (b) exposing the cancer cell to an effective amount of acombination of a procaspase activator compound and a second activeagent. Also provided is a method of treating a cancer cell, comprising(a) identifying a potential susceptibility to treatment of a cancer cellwith a procaspase activator compound; and (b) exposing said cancer cellto an effective amount of PAC-1 and a second active agent; wherein thePAC-1 is capable of activating at least one of procaspase-3 andprocaspase-7. Also provided is a method of inducing death in a cancercell (e.g., killing a cancer cell), comprising administering to a cancercell an active agent and a compound capable of activating a procaspase-3molecule of the cancer cell, such as PAC-1.

The invention further provides a medicament comprising an effectiveamount of the combination of PAC-1 and a second active agent. Themedicament can be used in a method of inducing apoptosis in a cell. Insome embodiments, the combination of compounds does not cross theblood-brain barrier to as extent that causes appreciable neurotoxiceffects in a patient. Methods of the invention include contacting one ormore cells with an effective amount of a combination of compoundsdescribed herein, in vivo or in vitro. The invention thus also providesmethods of treating a cell that include contacting a cell with aneffective amount of a combination of compounds described herein, andtreating a patient in need of cancer therapy with an effective amount ofa combination of compounds described herein.

As described herein, the invention provides methods of treating apatient that has tumor cells having elevated procaspase-3 levels. Themethods can include administering to a patient having tumor cells withelevated procaspase-3 levels a therapeutically effective amount of acombination of PAC-1 and a second active agent described herein, or acomposition thereof. The invention further provides methods of treatinga tumor cell having an elevated procaspase-3 level comprising exposingthe tumor cell to a therapeutically effective amount of a combination ofPAC-1 and a second active agent described herein, wherein the tumor cellis treated, killed, or inhibited from growing. The tumor or tumor cellscan be malignant tumor cells. In some embodiments, the tumor cells aremelanoma, colorectal, thyroid, lung, or ovarian cancer cells.

PAC-1 can be combined with a second active agent in a unitary dosageform for the administration to a patient. The combination therapy may beadministered as a simultaneous or sequential regimen. When administeredsequentially, the combination may be administered in two or moreadministrations.

The combination therapy may provide “synergy”, i.e. the effect achievedwhen the active ingredients used together is greater than the sum of theeffects that results from using the compounds separately. A synergisticeffect may be attained when PAC-1 and a second active agent are: (1)co-formulated and administered or delivered simultaneously in a combinedformulation; (2) delivered by alternation or in parallel as separateformulations; or (3) by some other regimen. When delivered inalternation therapy, a synergistic effect may be attained when thecompounds are administered or delivered sequentially, e.g. in separatetablets, pills or capsules, or by different injections in separatesyringes. In general, during alternation therapy, an effective dosage ofeach active ingredient can be administered sequentially, i.e. serially,whereas in combination therapy, effective dosages of two or more activeingredients are administered together. A synergistic anti-cancer effectdenotes an anti-cancer effect that is greater than the predicted purelyadditive effects of the individual compounds of the combination.Combination therapy is further described by U.S. Pat. No. 6,833,373(McKearn et al.), which includes additional active agents that can becombined with PAC-1, and additional types of cancer and other conditionsthat can be treated with PAC-1.

Accordingly, PAC-1 can be used in combination with the second activeagent for cancer treatment. PAC-1 may precede or follow the secondactive agent administration by intervals ranging from minutes to weeks.In embodiments where the second active agent and PAC-1 are appliedseparately to the cell, one would generally ensure that a significantperiod of time did not elapse between each delivery, such that the agentand PAC-1 would still be able to exert an advantageously combined effecton the cell. For example, in such instances, it is contemplated that onemay contact the cell, tissue or organism with the two modalitiessubstantially simultaneously (i.e., within less than about a fewminutes). In other aspects, the second active agent of the combinationmay be administered within about 1 minute, about 5 minutes, about 10minutes, about 20 minutes about 30 minutes, about 45 minutes, about 60minutes, about 2 hours, about 3 hours, about 4 hours, about 6 hours,about 8 hours, about 9 hours, about 12 hours, about 15 hours, about 18hours, about 21 hours, about 24 hours, about 28 hours, about 31 hours,about 35 hours, about 38 hours, about 42 hours, about 45 hours, or atabout 48 hours or more, prior to and/or after administering PAC-1. Incertain other embodiments, the second active agent may be administeredwithin about 1 day, about 2 days, about 3 days, about 4 days, about 5days, about 6 days, about 8 days, about 9 days, about 12 days, about 15days, about 16 days, about 18 days, about 20 days, or about 21 days,prior to and/or after administering PAC-1. In some situations, it may bedesirable to extend the time period for treatment significantly,however, where several weeks (e.g., about 1, about 2, about 3, about 4,about 6, or about 8 weeks or more) lapse between the respectiveadministrations.

Administration of the chemotherapeutic compositions of the invention toa patient will typically follow general protocols for the administrationof chemotherapeutics, taking into account the toxicity, if any. It isexpected that the treatment cycles would be repeated as necessary. Italso is contemplated that various standard therapies or adjunct cancertherapies, as well as surgical intervention, may be applied incombination with the described combinations. These therapies include butare not limited to chemotherapy, immunotherapy, gene therapy andsurgery.

Pharmaceutical Formulations

The compounds described herein can be used to prepare therapeuticpharmaceutical compositions, for example, by combining the compoundswith a pharmaceutically acceptable diluent, excipient, or carrier. Thecompounds may be added to a carrier in the form of a salt or solvate.For example, in cases where compounds are sufficiently basic or acidicto form stable nontoxic acid or base salts, administration of thecompounds as salts may be appropriate. Examples of pharmaceuticallyacceptable salts are organic acid addition salts formed with acids thatform a physiological acceptable anion, for example, tosylate,methanesulfonate, acetate, citrate, malonate, tartrate, succinate,benzoate, ascorbate, α-ketoglutarate, and β-glycerophosphate. Suitableinorganic salts may also be formed, including hydrochloride, halide,sulfate, nitrate, bicarbonate, and carbonate salts.

Pharmaceutically acceptable salts may be obtained using standardprocedures well known in the art, for example by reacting a sufficientlybasic compound such as an amine with a suitable acid to provide aphysiologically acceptable ionic compound. Alkali metal (for example,sodium, potassium or lithium) or alkaline earth metal (for example,calcium) salts of carboxylic acids can also be prepared by analogousmethods.

The compounds of the formulas described herein can be formulated aspharmaceutical compositions and administered to a mammalian host, suchas a human patient, in a variety of forms. The forms can be specificallyadapted to a chosen route of administration, e.g., oral or parenteraladministration, by intravenous, intramuscular, topical or subcutaneousroutes.

The compounds described herein may be systemically administered incombination with a pharmaceutically acceptable vehicle, such as an inertdiluent or an assimilable edible carrier. For oral administration,compounds can be enclosed in hard or soft shell gelatin capsules,compressed into tablets, or incorporated directly into the food of apatient's diet. Compounds may also be combined with one or moreexcipients and used in the form of ingestible tablets, buccal tablets,troches, capsules, elixirs, suspensions, syrups, wafers, and the like.Such compositions and preparations typically contain at least 0.1% ofactive compound. The percentage of the compositions and preparations canvary and may conveniently be from about 0.5% to about 60%, about 1% toabout 25%, or about 2% to about 10%, of the weight of a given unitdosage form. The amount of active compound in such therapeuticallyuseful compositions can be such that an effective dosage level can beobtained.

The tablets, troches, pills, capsules, and the like may also contain oneor more of the following: binders such as gum tragacanth, acacia, cornstarch or gelatin; excipients such as dicalcium phosphate; adisintegrating agent such as corn starch, potato starch, alginic acidand the like; and a lubricant such as magnesium stearate. A sweeteningagent such as sucrose, fructose, lactose or aspartame; or a flavoringagent such as peppermint, oil of wintergreen, or cherry flavoring, maybe added. When the unit dosage form is a capsule, it may contain, inaddition to materials of the above type, a liquid carrier, such as avegetable oil or a polyethylene glycol. Various other materials may bepresent as coatings or to otherwise modify the physical form of thesolid unit dosage form. For instance, tablets, pills, or capsules may becoated with gelatin, wax, shellac or sugar and the like. A syrup orelixir may contain the active compound, sucrose or fructose as asweetening agent, methyl and propyl parabens as preservatives, a dye andflavoring such as cherry or orange flavor. Any material used inpreparing any unit dosage form should be pharmaceutically acceptable andsubstantially non-toxic in the amounts employed. In addition, the activecompound may be incorporated into sustained-release preparations anddevices.

The active compound may be administered intravenously orintraperitoneally by infusion or injection. Solutions of the activecompound or its salts can be prepared in water, optionally mixed with anontoxic surfactant. Dispersions can be prepared in glycerol, liquidpolyethylene glycols, triacetin, or mixtures thereof, or in apharmaceutically acceptable oil. Under ordinary conditions of storageand use, preparations may contain a preservative to prevent the growthof microorganisms.

Pharmaceutical dosage forms suitable for injection or infusion caninclude sterile aqueous solutions, dispersions, or sterile powderscomprising the active ingredient adapted for the extemporaneouspreparation of sterile injectable or infusible solutions or dispersions,optionally encapsulated in liposomes. The ultimate dosage form should besterile, fluid and stable under the conditions of manufacture andstorage. The liquid carrier or vehicle can be a solvent or liquiddispersion medium comprising, for example, water, ethanol, a polyol (forexample, glycerol, propylene glycol, liquid polyethylene glycols, andthe like), vegetable oils, nontoxic glyceryl esters, and suitablemixtures thereof. The proper fluidity can be maintained, for example, bythe formation of liposomes, by the maintenance of the required particlesize in the case of dispersions, or by the use of surfactants. Theprevention of the action of microorganisms can be brought about byvarious antibacterial and/or antifungal agents, for example, parabens,chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, buffers, or sodium chloride. Prolonged absorption of theinjectable compositions can be brought about by agents delayingabsorption, for example, aluminum monostearate and/or gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in the appropriate solvent with variousother ingredients enumerated above, as required, optionally followed byfilter sterilization. In the case of sterile powders for the preparationof sterile injectable solutions, methods of preparation can includevacuum drying and freeze drying techniques, which yield a powder of theactive ingredient plus any additional desired ingredient present in thesolution.

Useful solid carriers include finely divided solids such as talc, clay,microcrystalline cellulose, silica, alumina, and the like. Useful liquidcarriers include water, dimethyl sulfoxide (DMSO), alcohols, glycols, orwater-alcohol/glycol blends, in which a compound can be dissolved ordispersed at effective levels, optionally with the aid of non-toxicsurfactants. Adjuvants such as fragrances and additional antimicrobialagents can be added to optimize the properties for a given use. Theresultant liquid compositions can be applied from absorbent pads, usedto impregnate bandages and other dressings, or sprayed onto the affectedarea using a pump-type or aerosol sprayer. Thickeners such as syntheticpolymers, fatty acids, fatty acid salts and esters, fatty alcohols,modified celluloses, or modified mineral materials can also be employedwith liquid carriers.

Useful dosages of the active agents described herein can be determinedby comparing their in vitro activity, and in vivo activity in animalmodels. Methods for the extrapolation of effective dosages in mice, andother animals, to humans are known to the art; for example, see U.S.Pat. No. 4,938,949 (Borch et al.). The amount of a compound, or anactive salt or derivative thereof, required for use in treatment willvary not only with the particular compound or salt selected but alsowith the route of administration, the nature of the condition beingtreated, and the age and condition of the patient, and will beultimately at the discretion of an attendant physician or clinician.

In general, however, a suitable dose of active agents will be in therange of from about 0.5 to about 100 mg/kg, e.g., from about 10 to about75 mg/kg, of body weight per day, such as 3 to about 50 mg per kilogrambody weight of the recipient per day, preferably in the range of 6 to 90mg/kg/day, most preferably in the range of 15 to 60 mg/kg/day. Thecompound can be conveniently formulated in unit dosage form; forexample, containing 5 mg to 1000 mg, conveniently 10 mg to 750 mg, mostconveniently, 50 mg to 500 mg of active ingredient per unit dosage form.In some embodiments, a PAC-1 dosage will be about 50-250 mg/kg, about75-150 mg/kg, or about 100 mg/kg. In various embodiments, the inhibitorof the BRAF gene or enzyme dosage will be about 0.5 mg/kg to about 25mg/kg, about 5 mg/kg to about 15 mg/kg, or about 10 mg/kg. MEK inhibitordosages can be of similar amounts to either of these active agents, orin about one-half to about one-tenth the amount of either of theseactive agents. In one embodiment, the invention provides a compositioncomprising an active agent or combination of active agents describedherein, formulated in one or more of such unit dosage forms.

The desired dose may conveniently be presented in a single dose or asdivided doses administered at appropriate intervals, for example, astwo, three, four or more sub-doses per day. The sub-dose itself may befurther divided, e.g., into a number of discrete loosely spacedadministrations; such as multiple inhalations from an insufflator or byoral administration.

The combination of active agents can be conveniently administered in aunit dosage form, for example, containing 100 to 5,000 mg/m², 300 to4,000 mg/m², 370 to 3,700 mg/m², 50 to 750 mg/m², or 750 to 4,000 mg/m²of active agent per unit dosage form. Each active agent, individually orin combination, can also be administered at about 1 mg/kg to about 250mg/kg, about 10 mg/kg to about 100 mg/kg, about 10 mg/kg to about 50mg/kg, about 50 mg/kg to about 100 mg/kg, about 10 mg/kg to about 50mg/kg, or about 10 mg/kg, about 25 mg/kg, about 50 mg/kg, about 75mg/kg, about 100 mg/kg, or about 150 mg/kg, or a range from any one ofthe aforementioned values to any other of the aforementioned values. Theactive agent can also be administered to a subject to provide asteady-state plasma concentration of the drugs, alone or in combination,of about 1 μmol/L to about 25 μmol/L, or about 10 μmol/L, or about 15μmol/L.

In some embodiments, the invention provides the active agent ineffective concentrations at about 10 nM to about 100 μM. In anotherembodiment, the effective concentrations are from about 200 nM to about50 μM, about 500 nM to about 40 μM, about 750 nM to about 25 μM, about 1μM to about 20 μM, or about 1 μM to about 10 μM. In another embodiment,the effective concentration is considered to be a value such as a 50%activity concentration in a direct procaspase activation assay, in acell apoptosis induction assay, or in an animal clinical therapeuticassessment. In one embodiment, such value is less than about 200 μM. Inanother embodiment, the value is less than about 10 μM but greater thanabout 10 nM. The desired dose may conveniently be presented in a singledose or as divided doses administered at appropriate intervals, forexample, as two, three, four or more sub-doses per day. The sub-doseitself may be further divided, e.g., into a number of discrete looselyspaced administrations.

The active agents described herein can be effective anti-tumor agentsand have higher potency and/or reduced toxicity as compared to theadministration of any single agent. The invention provides therapeuticmethods of treating cancer in a patient or subject, such as a mammal,which involve administering to a mammal having cancer an effectiveamount of a compound or composition described herein. A mammal includesa primate, human, rodent, canine, feline, bovine, ovine, equine, swine,caprine, bovine and the like. Cancer refers to any various type ofmalignant neoplasm, for example, colon cancer, breast cancer, melanoma,or leukemia, among others described herein, and in general ischaracterized by an undesirable cellular proliferation, e.g.,unregulated growth, lack of differentiation, local tissue invasion, andmetastasis.

The ability of a composition to treat cancer may be determined by usingassays well known to the art. For example, the design of treatmentprotocols, toxicity evaluation, data analysis, quantification of tumorcell kill, and the biological significance of the use of transplantabletumor screens are known. In addition, ability of a composition to treatcancer may be determined using the assays in the citations and patentdocuments cited herein.

The invention also provides prodrug forms of compounds. Any compoundthat will be converted in vivo to provide PAC-1 or another active agentrecited herein is a prodrug. Numerous methods of forming prodrugs arewell known in the art. Examples of prodrugs and methods of preparingthem are found, inter alia, in Design of Prodrugs, edited by H.Bundgaard, (Elsevier, 1985), Methods in Enzymology, Vol. 42, at pp.309-396, edited by K. Widder, et. al. (Academic Press, 1985); A Textbookof Drug Design and Development, edited by Krosgaard-Larsen and H.Bundgaard, Chapter 5, “Design and Application of Prodrugs,” by H.Bundgaard, at pp. 113-191, 1991); H. Bundgaard, Advanced Drug DeliveryReviews, Vol. 8, p. 1-38 (1992); H. Bundgaard, et al., Journal ofPharmaceutical Sciences, Vol. 77, p. 285 (1988); and Nogrady (1985)Medicinal Chemistry A Biochemical Approach, Oxford University Press, NewYork, pages 388-392).

Additionally, in some embodiments, PAC-1 can be exchanged for a PAC-1derivative or other inhibitor, such as a compound described in U.S. Pat.No. 7,632,972 (Hergenrother et al.), U.S. Pat. No. 8,778,945(Hergenrother et al.), or U.S. Pat. No. 8,916,705 (Hergenrother et al.),U.S. Patent Publication Nos. 2007/0049602 (Hergenrother et al.), U.S.application Ser. No. 12/597,287 (Hergenrother et al.), or InternationalPublication No. WO 2014/022858 (Hergenrother et al.), which areincorporated herein by reference. Useful compounds, methods, andtechniques for cancer therapy that can be used in combination with thedisclosure herein are described in the aforementioned documents, as wellas in U.S. Pat. No. 6,303,329 (Heinrikson et al.), U.S. Pat. No.6,403,765 (Alnemri), U.S. Pat. No. 6,878,743 (Choong et al.), and U.S.Pat. No. 7,041,784 (Wang et al.), and U.S. Patent Publication No.2004/0180828 (Shi).

Methods for performing the tests and evaluating cancer cell lines can becarried out as described by Putt et al., Nature Chemical Biology 2006,2(10), 543-550; Peterson et al., J. Mol. Biol. 2009, 388, 144-158; andPeterson et al., Cancer Res. 2010, 70(18), 7232-7241.

The following Examples are intended to illustrate the above inventionand should not be construed as to narrow its scope. One skilled in theart will readily recognize that the Examples suggest many other ways inwhich the invention could be practiced. It should be understood thatnumerous variations and modifications may be made while remaining withinthe scope of the invention.

EXAMPLES Example 1. The Combination of Vemurafenib and Procaspase-3Activation is Synergistic in Mutant BRAF Melanomas

The development of vemurafenib resistance limits the long-term efficacyof this drug for treatment of metastatic melanomas with the ^(V600E)BRAFmutation. Inhibition of downstream MAPK signaling with vemurafenibinduces apoptotic cell death mediated by caspase-3, suggesting thataddition of a procaspase-3 activator could enhance anticancer effects.Here we show that the combination of PAC-1, a procaspase-activatingcompound, and vemurafenib is highly synergistic in enhancing caspase-3activity and apoptotic cell death in melanoma cell lines harboring the^(6V00E)BRAF mutation. In vivo, the combination displays a favorablesafety profile in mice, and exerts significant antitumor effects. Wefurther demonstrate that addition of PAC-1 to the clinically usefulcombination of vemurafenib and an MEK inhibitor, trametinib, starklyenhances the caspase-3 activity and proapoptotic effect of thecombination. Moreover, addition of low concentration PAC-1 also delaysthe regrowth of cells following treatment with vemurafenib. Finally,PAC-1 remains potent against vemurafenib-resistant A375VR cells in cellculture and synergizes with vemurafenib to exert antitumor effects onA375VR cell growth in vivo. Collectively, our data indicate thatinhibition of MAPK signaling combined with concurrent procaspase-3activation is an effective strategy to enhance the antitumor activity ofvemurafenib and mitigate the development of resistance.

Here we report the synergistic activity of PAC-1+vemurafenib andPAC-1+vemurafenib+trametinib in enhancement of caspase-3 activity andapoptotic cell death in ^(V600E)BRAF melanoma. As a result of increasedapoptotic cell death, the PAC-1+vemurafenib combination inducessignificant reduction in tumor volume in a murine xenograft model of^(V600E)BRAF melanoma, well beyond the antitumor effects of theindividual agents. In addition, this enhancement of apoptotic death invemurafenib-sensitive melanoma by the addition of PAC-1 significantlydelays the regrowth of cells after exposure to vemurafenib. Finally,PAC-1 remains effective in vemurafenib-resistant A375VR cells in cultureand synergizes with vemurafenib to retard tumor growth of these cells invivo, demonstrating utility of this combination in melanomas that haveprogressed beyond BRAF-inhibitor treatment, for which few options fortreatment are currently available.

The combination of PAC-1 and vemurafenib enhances apoptosis in cellswith the ^(V600E)BRAF mutation.

In a panel of nine cell lines of diverse origins and BRAF mutationalstatus, vemurafenib is potent (IC₅₀ values between 200-550 nM) only incell lines harboring the ^(V600E)BRAF mutation, consistent withpreviously reported values (FIG. 1A). Evaluation of PAC-1 in the samepanel of cell lines shows that PAC-1 retains similar activity in allcell lines (IC₅₀ values between 1-4 regardless of BRAF mutational status(FIG. 1A). The ability of the combination of PAC-1+vemurafenib to induceapoptotic cell death was then assessed in these cell lines. Underconditions (24 h incubation with compounds) where neither vemurafenibnor PAC-1 induced significant apoptotic death (≤10%) as single agents,the PAC-1+vemurafenib combination induces significant apoptosis (20-45%)in cell lines with the ^(V600E)BRAF mutation (FIG. 1B). A similar trendwas also observed when a lower concentration of vemurafenib (0.5 μM) wasevaluated in combination with PAC-1 in ^(6V00E)BRAF cell lines (FIG.1C). However, the PAC-1+vemurafenib combination does not inducesynergistic apoptosis in cell lines with wild-type BRAF (FIG. 1B).

PAC-1 and vemurafenib synergize to enhance caspase-3 activity andapoptosis in A375, SK-MEL-5 and UACC-62 cells.

In order to more broadly explore the observed synergy, apoptotic deathwas assessed in three human ^(V600E)BRAF melanoma cell lines treatedwith a matrix of concentrations of PAC-1 and vemurafenib that induceminimal apoptosis as single agents. In these experiments, largeincreases in the populations of apoptotic cells (beyond the additiveeffect of single agents alone) were observed in A375 (FIG. 2A), SK-MEL-5(FIG. 7A) and UACC-62 (FIG. 8A). To quantify the synergy of this drugcombination, combination indices (CI) were calculated. A drugcombination that is synergistic will have a CI value less than 1, whilea value of 1 reflects an additive effect (Chou, Pharmacol Rev 2006;58:621-81). 93% of the calculated CI values are less than 1 (A375 inFIG. 2B, SK-MEL-5 in FIG. 7B and UACC-62 in FIG. 8B), indicatingsynergism for the combination across all three cell lines tested.

To assess if the increase in apoptosis was a result of increasedactivation of executioner procaspases, caspase-3/-7 enzymatic activitywas evaluated in A375 cells (after lysis) using a fluorogenic substrate.In A375 cells treated with vemurafenib or PAC-1 alone (at the sameconcentrations used in FIG. 1B), negligible increases in caspase-3activity were observed at these time points and concentrations (FIG.2C). However, when A375 cells were treated with PAC-1 and vemurafenib, asignificant increase in caspase-3 activity was observed as early as 7 hpost-treatment (FIG. 2C). In Western blot analyses, neither of thesingle agents had an effect on PARP-1 cleavage at these time points andconcentrations; however, the combination resulted in significant cleavedPARP-1 (FIG. 2D), a result of the increased caspase-3/-7 activity incells treated with the PAC-1+vemurafenib combination. After treatmentwith the combination for 24 h, near-complete cleavage of PARP-1 wasobserved in A375 cells (FIG. 2D). Similar results for the caspase-3/-7activity assay and cleavage of PARP-1 were also observed in SK-MEL-5(FIGS. 7C and 7D) and UACC-62 cells (FIGS. 8C and 8D).

The PAC-1 derivative PAC-1a lacks the zinc chelating motif and thus doesnot activate procaspase-3 or induce apoptosis.

Use of PAC-1a in combination with vemurafenib did not result in asignificant increase in the proportion of cells undergoing apoptosis inA375, SK-MEL-5 or UACC-62 cells (FIG. 9A-C). This result is alsoconsistent with the absence of increased PARP-1 cleavage in cellstreated with the PAC-1a and vemurafenib combination (FIG. 9D),indicating that the cells did not undergo apoptotic death.

Inhibition of ERK1/2 Phosphorylation and Activation of Procaspase-3 areRequired to Enhance Apoptotic Cell Death.

Consistent with the data in FIG. 1B, no enhancement in caspase-3activity or PARP-1 cleavage were observed in two ^(WT)BRAF cell lineswhen treated with the combination of PAC-1+vemurafenib (FIG. 1A-C). Thelack of PAC-1+vemurafenib synergy in cell lines harboring ^(WT)BRAFsuggests that inhibition of ERK1/2 and activation of procaspase-3 areboth required to induce the dramatic enhancement of apoptotic celldeath. Indeed, after 24 h of treatment with vemurafenib, inhibition ofERK1/2 phosphorylation was not observed in ^(WT)BRAF cell lines even athigh concentrations (30 μM) of vemurafenib (FIGS. 1B and 1C). Thisobservation is consistent with previous reports where vemurafenib doesnot inhibit ERK1/2 phosphorylation in ^(WT)BRAF cells, but paradoxicallyactivates it.

To further investigate this, A375 (harboring ^(6V00E)BRAF) cells weretreated with PAC-1, vemurafenib, or the combination and probed for thepresence of cleaved PARP-1 and ERK1/2 phosphorylation. After 24 h,phospho-ERK1/2 bands were not observed in cells treated with vemurafenib(at 0.5 and 1.0 μM) and the combination (FIG. 2E). However, significantincreases in the amount of cleaved PARP-1 were only observed in cellstreated with both PAC-1 and vemurafenib (FIG. 2E). Similar results werealso observed in SK-MEL-5 (FIG. 7E) and UACC-62 cells (FIG. 8E). At lowconcentrations of vemurafenib (0.1 and 0.25 where incomplete inhibitionof ERK1/2 phosphorylation was observed, slight increase in PARP-1cleavage over that single agent effects was also observed (FIG. 2E).This result suggests that even with incomplete inhibition of ERK1/2phosphorylation, procaspase-3 activation, which is downstream of ERK1/2signaling, can be enhanced with the addition of PAC-1 to vemurafenibtreatments. Taken together, the data show that procaspase-3 activationvia PAC-1 dramatically enhances the proapoptotic effect of vemurafenibin cell lines with ^(6V00E)BRAF mutation.

Addition of PAC-1 to Vemurafenib and Trametinib Enhances Caspase-3Activity and Apoptosis.

Addition of a MEK1/2 inhibitor, such as trametinib, is widely used inthe clinic to enhance the efficacy of vemurafenib in ^(V600E)BRAFmelanomas. To explore the effect of PAC-1 with this combination, cellswere treated with vemurafenib+trametinib, in the presence or absence ofPAC-1, and apoptosis was assessed. In both A375 and UACC-62 cell lines,vemurafenib+trametinib co-treatment led to mere additive increases inthe population of apoptotic cells (FIG. 3A). In contrast, the additionof PAC-1 led to a large increase in the population of apoptotic cells,beyond the additive effect of single agents alone (FIG. 3A).Vemurafenib+trametinib co-treatment did not lead to PARP-1 cleavage,while addition of PAC-1 led to near quantitative cleavage of PARP-1(FIG. 3B). To explore if the increased apoptotic cell death in thepresence of PAC-1 is a result of enhanced enzymatic activity ofexecutioner caspases, the caspase-3/-7 activity of A375 and UACC-62cells treated with vemurafenib+trametinib, plus or minus PAC-1, wasassessed. Again, a dramatic increase in caspase-3/-7 activity wasobserved when PAC-1 was included, an effect that was absent withoutaddition of PAC-1 (FIG. 3C).

The Combination of Vemurafenib and PAC-1 Significantly Reduces TumorBurden in an A375 Xenograft Model.

To determine the antitumor effect of the PAC-1+vemurafenib combinationin vivo, an A375 xenograft model (Yadav et al., Mol Cancer Ther 2014;13:2253-63) was used. In this model, nude mice were inoculatedsubcutaneously with A375 cells, and after allowing the tumors to grow,mice were randomized based upon tumor volume into four groups[F=0.03<F_(critical)(3.01)] and dosed with PAC-1, vemurafenib, or thecombination for 15 days. Treatment with PAC-1 alone led to minimalreduction in tumor mass and volume compared to untreated control mice(FIGS. 4A and 4B). Mice dosed with vemurafenib alone experienced amoderate reduction (53%; p=0.04) in tumor volume and mass compared tocontrol (FIGS. 4A and 4B), with 3 out of 8 mice having comparable tumormass as the control mice (FIG. 4B). In contrast, mice treated with thecombination of PAC-1 and vemurafenib had significantly smaller tumorburden compared to control mice (FIG. 4A, 4B and FIG. 11). In thesemice, a 78% reduction in tumor volume was observed (FIG. 4A, p=0.0008vs. control), with 6 out of 8 mice having tumors less than 0.2 g in mass(FIG. 4B), indicating that addition of PAC-1 enhances the antitumoreffects of vemurafenib in vivo and reduces the variability in responseto treatment.

Examination of procaspase-3 levels in the tumor samples by Western blotshowed an appreciable and consistent reduction in the amount ofprocaspase-3 only in tumor samples derived from mice that received thecombination treatment, versus variable responses for the other dosinggroups (FIGS. 4C and 4D). Using immunohistochemical staining, asignificant reduction in the percentage of Ki-67 expressing cells intumors treated with PAC-1+vemurafenib was observed (FIG. 4E), indicatingthat the PAC-1+vemurafenib combination was capable of not onlyamplifying procaspase-3 activation, but also attenuating cellproliferation. Finally, in mice treated with PAC-1+vemurafenib, nohematological toxicities were observed (Table 1), indicating a favorablesafety profile for the combination. Taken together, the in vivo data areconsistent with the cell culture results showing that the synergy ofPAC-1+vemurafenib leads to increase in caspase-3 activity and inductionof apoptotic cell death, as well as reduction in cell proliferation.

TABLE 1 Hematologic and biochemical toxicity of PAC-1 and vemurafenib.Average data from 4 mice treated with 100 mg/kg PAC-1 once-a-day and 10mg/kg vemurafenib twice-a-day for 15 days. No clinically significantevidence for myelosuppression, renal injury, or hepatic toxicity wasidentified. Blood chemistry Ave ± SEM Normal Range¹ Creatinine (mg/dL)0.20 ± 0.04 0.2-0.4 BUN (Urea) (mg/dL) 32.3 ± 1.0  11-39 Total Protein(g/dL) 4.7 ± 0.1 4.8-6.6 Albumin (g/dL) 2.2 ± 0.1 2.8-4.0 Globulin(g/dL) 2.5 ± 0.1 Calcium (mg/dL) 9.2 ± 0.2  9.5-12.1 Phosphorous (mg/dL)10.8 ± 0.5   8.0-15.5 Sodium (mmol/L) 161.0 ± 0.8  140.7-165.1 Potassium(mmol/L) 7.9 ± 0.2  7.0-10.8 Chloride (mmol/L) 119.0 ± 0.9  108.8-133.2Glucose (mg/dL) 182.3 ± 12.5  149-271 Alkaline Phos Total (U/L) 70.8 ±9.0   76-301 ALT (SGPT) (U/L) 50.5 ± 4.6   31-115 Total Bilirubin(mg/dL) 0.3 ± 0.1 0.2-0.5 Cholesterol total (mg/dL) 111.5 ± 4.4   98-202Platelet Estimate* (10³/μL) 229.3 ± 7.5   376-1796 WBC Estimate (10³/μL)3.2 ± 0.4  1.4-10.3 Seg % 31.5 ± 8.7  14.0-54.7 Lymph % 61.5 ± 9.6 23.6-79.3 *Platelet cell counts were low because platelet clumps wereobserved. ¹Normal range values were obtained from Charles River forfemale NU/NU mice between 8 to 10 weeks of age.

Long Term Treatment with PAC-1 Prevents Cell Regrowth, and Addition ofPAC-1 to Vemurafenib Delays the Onset of Cell Regrowth.

The E_(max) of vemurafenib (the percent cell death induced by highconcentrations of compound) in A375 cells is 96.8±0.3% after 5 days(FIG. 5A), indicating that ˜3% of A375 cells are insensitive tovemurafenib. Under the same conditions, PAC-1 has an E_(max) of99.4±0.7% (FIG. 5A), indicating that PAC-1 kills A375 cellsquantitatively, with very few insensitive cells. We thereforehypothesized that long term treatment with vemurafenib would lead tore-growth of cancer cells, while treatment with PAC-1 should preventre-growth. To investigate this hypothesis, A375 and SK-MEL-5 cells wereplated at low densities and treated continuously with PAC-1 (4 μM) orvemurafenib (10 μM) for up to 30 days. In A375 and SK-MEL-5 cellstreated with vemurafenib, regrowth of cells was observed in as early as20 days (FIG. 5B). However, in wells treated with PAC-1, no regrowth wasobserved even after 30 days (FIG. 5B). Thus, consistent with the higherE_(max) value, PAC-1 is able to quantitatively kill cells therebypreventing regrowth.

To investigate if addition of low concentrations of PAC-1 could combinewith vemurafenib to prevent cancer cell re-growth, A375 and UACC-62cells were plated at low densities in 96-well plates and treatedcontinuously with PAC-1 (1 μM), vemurafenib (5 μM or 10 μM), or thecombination for up to 20 days. After 5 days, treatment with PAC-1,vemurafenib, or the combination each resulted in significant reductionin cell number compared to the control (A375: FIGS. 5C and 5D; UACC-62:FIGS. 12A and 12B). On day 10, there is no observable difference betweenthe PAC-1 treated wells and the control. In wells treated with 5 μM or10 μM vemurafenib, cell death was 89.4±1.4% and 93.2±1.1%, respectively.However, in wells where A375 cells were treated with 1 μM PAC-1 and 5 μMor 10 μM vemurafenib, increased cell death was observed, 96.1±1.0% and97.9±0.7% respectively. Consequent to achieving more complete celldeath, a smaller proportion of cells remain in wells treated with bothPAC-1 and vemurafenib. After 20 days of treatment, significant regrowthof colonies was observed in vemurafenib-only treated wells but not inwells receiving the co-treatment (A375: FIGS. 5C and 5D; UACC-62: FIGS.12A and 12B). This result indicates that the more complete cell deathinduced by co-treating cells with PAC-1 and vemurafenib is effective indelaying the regrowth of A375 and UACC-62.

PAC-1 synergizes with vemurafenib in vemurafenib-resistant melanoma invivo.

To assess if PAC-1 remains active in a cell line that has acquiredresistance to vemurafenib, a vemurafenib-resistant A375VR cell line wasgenerated by growing A375 parental cell line in sequentially higherconcentrations of vemurafenib (0.5 μM to 1.0 μM) for 2 months. Todetermine the mechanism of resistance of A375VR, genes for MEK1/2, NRASand AKT were sequenced, but no commonly reported mutations that wouldconfer resistance were found (Rizos et al., Clinical Cancer Research2014; 20:1965-77). Similarly, splice variant of the ^(6V00E)BRAF mRNAwas also not observed. Through qPCR, A375VR cells have approximately3-fold higher levels of MDR1 mRNA compared to A375. However, compared toup to 1000-fold higher levels of MDR1 mRNA in ovarian cells resistant todoxorubicin or cisplatin, the level of MDR1 mRNA overexpression isconsidered low, indicating that resistance is unlikely due to dramaticupregulation of MDR phenotype.

Vemurafenib kills the A375VR cell line with a 5-day IC₅₀ value of 1.5μM, 12-fold less potent compared to the sensitivity of the parental A375(FIG. 6A). Moreover, the vemurafenib E_(max) for A375VR is 79±6.3%,which is 14% lower than the parental A375 cell line. While treatment ofparental A375 cells with vemurafenib (0.5 or 1 μM) for 2 h results incomplete inhibition of ERK1/2 phosphorylation, this effect is notobserved in A375VR, consistent with resistance of A375VR to vemurafeniband continued MAPK signaling (FIG. 6B). In contrast, PAC-1 retainsactivity against A375VR with an IC₅₀ value of 2.4 μM (vs 1.2 μM for theparental cell line, FIG. 6C) and a similar E_(max). We hypothesized thatdespite the inability of vemurafenib to inhibit ERK1/2 phosphorylationand MAPK signaling in the resistant A375VR cell line, the combinationmight retain partial capacity to exert a synergistic effect based on thePARP-1 cleavage observed for PAC-1+vemurafenib treatment, even underconditions of incomplete inhibition of ERK1/2 phosphorylation (FIG. 2E).To investigate if PAC-1 can re-sensitize A375VR cells tovemurafenib-induced apoptosis, A375VR cells were treated with PAC-1 incombination with low concentrations of vemurafenib. This combinationtreatment led to an increase in the proportion of cells undergoingapoptosis (FIG. 13A, 13C), indicating that the addition of PAC-1 canbypass the resistance mechanism of A375VR to vemurafenib. This effectwas abolished when inactive variant PAC-1a was used (FIG. 13C). ThePAC-1+vemurafenib combination was synergistic, inducing an average of7.5% higher population of apoptotic cells than predicted by the Blissindependence model (Bliss, Ann Appl Biol 1939; 26:585-615) (FIGS. 13Aand 13B). Finally, to determine if PAC-1 can synergize with vemurafenibin vivo, A375VR cells were implanted subcutaneously in nude mice, andthe mice were dosed daily for 15 days with vemurafenib (10 mg/kg), PAC-1(100 mg/kg) or the combination. Treatment with vemurafenib or PAC-1alone does not exert any antitumor affect in this in vivo model, whiletreatment with combination led to significant reduction in tumor volumecompared to the untreated control (FIG. 6D).

Discussion.

Given that the aberrations in the apoptotic signaling cascades inmelanoma cells are upstream of the activation of procaspase-3, smallmolecules that directly activate procaspase-3 can induce apoptosis bybypassing the defective apoptotic circuitry. Activation of procaspase-3with PAC-1 has been shown previously to have single agent efficacyagainst melanoma cells in culture (Wang et al., Mol Oncol 2014;8:1640-52; Peterson et al., Cancer Res 2010; 70:7232-41; Putt et al.,Nat Chem Biol 2006; 2:543-50), and now we show that PAC-1+vemurafenib,or PAC-1+vemurafenib+trametinib, are powerfully synergistic in theinduction of caspase-3 activity and apoptotic cell death in melanomaswith ^(6V00E)BRAF mutation. Besides melanomas, the ^(V600E)BRAF mutationhas been reported in several other cancers including Erdheim-Chesterdisease (ECD) (54%), Langerhans'-cell histiocytosis (LCH) (57%),non-small-cell lung cancer (NSCLC) (1.5%) and hairy-cell leukemia(100%). In two recent Phase II trials, efficacy of vemurafenib inseveral non-melanoma cancers harboring the ^(V600E)BRAF mutation wasreported, with promising results seen in patients with NSCLC, ECD, LCHand refractory hairy-cell leukemia. Given this clinical data and ourcurrent work showing potent synergy between PAC-1, vemurafenib, andtrametinib in ^(6V00E)BRAF melanomas, these PAC-1/drug combinations canhave efficacy in other malignancies harboring the ^(V600E)BRAF mutation.

The E_(max) parameter is a useful metric to assess the ability of acompound to quantitatively kill cancer cells in culture (Fallahi-Sichaniet al., Nat Chem Biol 2013; 9:708-14). E_(max) values less than 100%imply heterogeneity in the ability of the drug to kill the cancer cellpopulation. Here we show that vemurafenib has an E_(max) of ˜97% in^(6V00E)BRAF mutant A375 cells, but the E_(max) value for PAC-1approaches 100%. Because of this, no regrowth of A375 or SK-MEL-5 cellsis observed in long-term experiments with PAC-1. However, extensiveregrowth was observed in A375, UACC-62 and SK-MEL-5 cells treated onlywith vemurafenib for 20 days. With the addition of a low concentrationof PAC-1 (1 μM) to vemurafenib, little to no regrowth was observed incells. These results indicate that addition of low concentrations ofPAC-1 (1 μM, a PAC-1 concentration that is readily achieved in vivo(Lucas et al., Invest New Drugs 2011; 29:901-11)) can be effectiveclinically in delaying resistance. The significant increase in caspase-3activity, followed by massive induction of apoptosis early on during thecombination treatment, likely kills off a large proportion of the cellsthat were initially insensitive to vemurafenib. Consequently, there is asignificantly smaller residual population of cells that are unaffectedby the treatment, crucial to delaying the regrowth of cells.

Currently, few options exist for patients who have developedvemurafenib-resistant melanomas. The MEK1/2 inhibitor, trametinib,though approved for melanomas with ^(V600E)BRAF mutation, exerts limitedactivity in combination with BRAF inhibitor in patients who have failedprior therapy (Kim et al., J Clin Oncol 2013; 31:482-89). Our resultsshow that PAC-1 still synergizes with vemurafenib to exert antitumoreffects in vemurafenib-resistant tumors. Therefore, addition of PAC-1might be a viable and alternative therapeutic option for patients whosemelanomas have progressed after vemurafenib treatment. ThePAC-1+vemurafenib combination is well tolerated, has a good safetyprofile and exhibits significant antitumor effects in vivo. PAC-1 iscurrently in a Phase I clinical trial (NCT02355535), and bothvemurafenib and trametinib are approved first-line treatment for^(V600E)BRAF melanoma. There is thus a clear path to translate thepreclinical demonstration of synergy described in this work to clinicaltrials where this novel combination can be assessed in human patientswith cancers harboring the ^(V600E)BRAF mutation.

Materials and Methods

Cell culture and reagents. A375 (CRL-1619) and CHL-1 (CRL-9446) werepurchased from ATCC on Nov. 5, 2014 and Nov. 18, 2014 respectively.A375SM was provided by Prof. Isiah Fidler (MD Anderson, Tex.) on Oct.30, 2014. All cell lines except B16-F10, H460, and HCT 116 were culturedin DMEM supplemented with 10% FBS (Gemini). B16-F10, H460, and HCT 116were cultured in RPMI with 10% FBS. Vemurafenib, trametinib and AnnexinV-FITC (10040-02) were purchased from LC Laboratories, MedChemExpress,and SouthernBiotech respectively. The following antibodies werepurchased from Cell Signalling Technology: anti-PARP-1 (9542),anti-caspase-3 (9662), anti-β-actin (4967), anti-phospho-ERK1/2(Thr202/Tyr204) (4370), anti-ERK1/2 (4695) and anti-rabbit IgG HRPlinked (7074). Anti-cleaved-PARP-1 (ab32561) antibody was purchased fromEpitomics. PAC-1 and PAC-1a were synthesized as previously reported(Putt et al., Nat Chem Biol 2006; 2:543-50).

Cell line authentication.

All human cell lines (A375, A375SM, CHL-1, H460, HCT 116, MIA PaCa-2,SK-MEL-5, and UACC-62) have been authenticated using the PowerPlex16HSAssay (Promega): 15 Autosomal Loci, X/Y at the University of ArizonaGenetics Core. The results of the test and pherograms were recorded.Mycoplasma testing has been performed for the A375 cell line using theMycoplasma detect PCR at the University of Illinois VeterinaryDiagnostic Lab.

Cellular proliferation assays.

1000-2000 cells were seeded per well in a 96-well plate and allowed toadhere before DMSO solutions of PAC-1 or vemurafenib were added to eachwell. Proliferation was assessed by the sulforhodamine B (SRB) assay.

Annexin V/PI flow cytometry analysis.

70,000 cells were seeded in 12-well plates and allowed to adhere beforeaddition of compounds. Cells were treated with compounds for 24 h at 37°C., after which they were harvested and resuspended in 450 μL of coldbuffer (10 mM HEPES, 140 mM NaCl, 2.5 mM CaCl₂ pH 7.4) premixed withAnnexin V-FITC and PI (0.55 μg/mL) dyes. Samples were analyzed on a BDBiosciences LSR II flow cytometer and data analysis was performed usingFCS Express V3-2.

Caspase-3/7 activity assay.

5,000-8,000 cells were plated in 96 well plates and allowed to adhere.Cells were treated with 1 μM of staurosporine for 24 h or with 13 μM ofraptinal (Palchaudhuri et al., Cell Rep 2015; 13:2027-36) for 3 h aspositive control, DMSO as negative control and indicated concentrationsof PAC-1 and vemurafenib for 0, 2, 4, 7, 10, 12, 16, 20 or 24 h. Plateswere then assessed for caspase-3/7 activity via addition of bifunctionallysis and activity buffer (200 mM HEPES, 400 mM NaCl, 40 mM DTT, 0.4 mMEDTA, 1% Triton-X, pH 7.4) with 20 μM of Ac-DEVD-AFC (Cayman Chemicals)as the fluorogenic substrate (λ_(ex)=400 nm, Xem=505 nm). Plates werepre-incubated at 37° C. at 30 min in the Synergy multi-mode reader(BioTek) then read for 30 min at 3 min intervals. The slopes for eachwell were calculated. Activity is expresses as normalized to minimal andmaximal activity observed within the assay.

In vitro resistance assay.

800 A375 or UACC-62 cells were plated in 96-well plates and allowed toattach overnight. The next day, vemurafenib (5 or 10 μM) or PAC-1 (1 μM)were treated in six technical replicates for 5, 10 and 20 days. Freshmedia and compounds were added every 2-3 days for the duration of thestudy. At the end of 5, 10 or 20 days, the wells were fixed with 10%cold trichloroacetic acid for 1 h at 4° C. The wells were then washed,allowed to dry and stained with 0.5% SRB dye for 30 min at roomtemperature. The wells were then washed with 0.1% acetic acid andallowed to dry. At this point, images of the plates were taken withGelDoc XR (BioRad). Finally, 200 μL of 10 mM Tris base (pH >10.4) wasadded into well and the absorbance at 510 nm were read using SpectraMaxPlus (Molecular Devices). The absorbance at 510 nm is plotted againstthe days post treatment as an indication of cell proliferation over thetime course of the experiment.

Immunoblotting.

Cells and tumor tissues were lysed using RIPA buffer containingphosphatase and protease inhibitor cocktail (Calbiochem). The proteinconcentration of each sample was determined by the BCA assay (Pierce).Cell lysates containing 20 μg of protein was loaded into each lane of4-20% gradient gels (BioRad) for SDS-PAGE. Proteins were transferredonto PDVF membrane for Western blot analysis.

PCR and sequencing.

A375 and A375VR cells were lysed and RNA extracted using the RNeasy kit(Qiagen). 900 ng of RNA was used for reverse transcription reactionusing iScript cDNA synthesis kit (BioRad). qPCR reactions were ran onthe 7900HT fast real-time PCR system (Applied Biosystems). Regular PCRreactions were ran using the MyFi Mix PCR kit (Bioline) for 35 cyclesand ran on a 1% agarose gel. Target amplicons were gel extracted andsequenced at the UIUC core sequencing facility. Primers used can befound in the following table.

Primer sequences used to characterize vemurafenib-resistant A375VR.

MDR1 F (SEQ ID NO: 1) ACACCATGGGGAAGGTGAAG MDR1 R (SEQ ID NO: 2)GTGACCAGGCGCCCAATA GAPDH F (SEQ ID NO: 3) ACACCATGGGGAAGGTGAAG GAPDH R(SEQ ID NO: 4) GTGACCAGGCGCCCAATA BRAF F (SEQ ID NO: 5)GGCTCTCGGTTATAAGATGGC BRAF R (SEQ ID NO: 6) ACAGGAAACGCACCATATCCMEK1 Amp F (SEQ ID NO: 7) CGTTACCCGGGTCCAAAATG MEK1 Amp R (SEQ ID NO: 8)CTTTGTCACAGGTGAAATGC MEK1 Seq F (SEQ ID NO: 9) CATGGATGGAGGTTCTCTGGMEK1 Seq R (SEQ ID NO: 10) AGGGCTTGACATCTCTGTGC MEK2 Amp F(SEQ ID NO: 11) CTCCCGGCCCGCCCCCTATG MEK2 Amp R (SEQ ID NO: 12)GTGGAGGCGCCAGCCTGTCC MEK2 Seq F (SEQ ID NO: 13) GTCAGCATCGCGGTTCTCCMEK2 Seq R (SEQ ID NO: 14) TCACCCCGAAGTCACACAG NRAS F (SEQ ID NO: 15)AGCTTGAGGTTCTTGCTGGT NRAS R (SEQ ID NO: 16) TCAGGACCAGGGTGTCAGTG AKT1 F(SEQ ID NO: 17) AGCGCCAGCCTGAGAGGA AKT1 Amp R (SEQ ID NO: 18)TCTCCATCCCTCCAAGCTAT AKT1 Seq R (SEQ ID NO: 19) GACAGGTGGAAGAACAGCT

A375 and A375VR xenograft model.

All animal studies were performed in accordance with UIUC IACUCguidelines (protocol no. 14292). 0.1 mL of A375 or A375VR in 1:1DMEM:matrigel (Corning) was injected into the right flank of 6-7 (A375)or 5 (A375VR) week old female athymic nude mice (Charles River). In theboth models, the mice were randomized into four groups: control, 100mg/kg PAC-1, 10 mg/kg vemurafenib, and the combination of 100 mg/kgPAC-1 and 10 mg/kg vemurafenib (n=8). Initial tumor volume measurementswere taken and dosing was initiated for a period of 15 days. Vemurafenibwas formulated as 5% DMSO in 1% methyl cellulose and given twice dailyby oral gavage (p.o.). PAC-1 was formulated in 200 mg/mLhydroxypropyl-β-cyclodextrin at pH 5.5 and given by intraperitoneal(i.p.) injection. Tumor length and width measurements were taken threetimes a week and volume was calculated as 0.52*L*W². At the end of thestudy, the mice were euthanized and tumors were excised. The tumors wereweighed and used for Western blot and immunohistochemistry.

Immunohistochemistry of A375 tumors and quantification of Ki-67 index.

Immunohistochemistry (IHC) was performed on 4 μm-thick formalin-fixedparaffin-embedded A375 tumors after H&E staining confirmed the presenceof a neoplastic cell population along with adequate tissue integrity.Antibody against Ki-67 (Biocare Medical #CRM325) was used for IHC andstaining was visualized using the IntelliPATH FLX DAB chromogen kit(Biocare Medical #IPK 5010 G80). Human tonsil was used as the positivecontrol tissue. Polymer negative control serum (mouse and rabbit)(Biocare Medical #NC499) was substituted for the primary antibody as anegative control. For quantification of Ki-67 index, 2000 neoplasticcells were counted and the percentage of positive cells was calculated.In tumors too small to quantify 2000 cells, the maximal number ofneoplastic cells were counted. All slides were reviewed by a singleveterinary pathologist.

Example 2. Pharmaceutical Dosage Forms

The following formulations illustrate representative pharmaceuticaldosage forms that may be used for the therapeutic or prophylacticadministration of the combination compounds described herein (e.g.,PAC-1 and the second active agent), or pharmaceutically acceptable saltsor solvates thereof (hereinafter referred to as ‘Compounds X’, which canbe one active agent or a combination of two active agents):

(i) Tablet 1 mg/tablet ‘Compounds X’ 200.0 Lactose 77.5 Povidone 15.0Croscarmellose sodium 12.0 Microcrystalline cellulose 92.5 Magnesiumstearate 3.0 400.0

(ii) Tablet 2 mg/tablet ‘Compounds X’ 120.0 Microcrystalline cellulose410.0 Starch 50.0 Sodium starch glycolate 15.0 Magnesium stearate 5.0600.0

(iii) Capsule mg/capsule ‘Compounds X’ 110.0 Colloidal silicon dioxide1.5 Lactose 465.5 Pregelatinized starch 120.0 Magnesium stearate 3.0700.0

(iv) Injection 1 (1 mg/mL) mg/mL ‘Compounds X’ 1.0 Dibasic sodiumphosphate 12.0 Monobasic sodium phosphate 0.7 Sodium chloride 4.5 1.0NSodium hydroxide solution q.s. (pH adjustment to 7.0-7.5) Water forinjection q.s. ad 1 mL

(v) Injection 2 (10 mg/mL) mg/mL ‘Compounds X’ 10.0 Monobasic sodiumphosphate 0.3 Dibasic sodium phosphate 1.1 Polyethylene glycol 400 200.00.1N Sodium hydroxide solution q.s. (pH adjustment to 7.0-7.5) Water forinjection q.s. ad 1 mL

(vi) Aerosol mg/can ‘Compounds X’ 20 Oleic acid 10Trichloromonofluoromethane 5,000 Dichlorodifluoromethane 10,000Dichlorotetrafluoroethane 5,000

These formulations may be prepared by conventional procedures well knownin the pharmaceutical art. It will be appreciated that the abovepharmaceutical compositions may be varied according to well-knownpharmaceutical techniques to accommodate differing amounts and types ofactive ingredient(s) ‘Compounds X’. Aerosol formulation (vi) may be usedin conjunction with a standard, metered dose aerosol dispenser.Additionally, the specific ingredients and proportions are forillustrative purposes. Ingredients may be exchanged for suitableequivalents (e.g., components described above) and proportions may bevaried, according to the desired properties of the dosage form ofinterest.

Example 3. Tablet Forms

The following formulation illustrates representative pharmaceuticaldosage forms that may be used for the therapeutic or prophylacticadministration of the combination compounds described herein (e.g.,PAC-1 and the second active agent), or pharmaceutically acceptable saltsor solvates thereof:

(i) Tablet A mg/tablet PAC-1 250.0 Microcrystalline cellulose 127.5Mannitol 50.0 Sodium starch glycolate 50.0 Fumed silica 2.5Hydroxypropyl cellulose 15.0 Sodium stearyl fumarate 5.0 500.0

(ii) Tablet B mg/tablet Second agent 250.0 Microcrystalline cellulose127.5 Mannitol 50.0 Sodium starch glycolate 50.0 Fumed silica 2.5Hydroxypropyl cellulose 15.0 Sodium stearyl fumarate 5.0 500.0The second agent can be, for example, vemurafenib, dabrafenib,BMS-908662 (also known as XL281), encorafenib (LGX818), PLX3603(R05212054), or RAF265(1-methyl-5-[2-[5-(trifluoromethyl)-1H-imidazol-2-yl]pyridin-4-yl]oxy-N-[4-(trifluoromethyl)phenyl]-benzimidazol-2-amine).The second agent can also be a MEK inhibitor, or a combination of a MEKinhibitor and one of the aforementioned actives. Furthermore, a thirdpharmaceutical dosage form similar to Tablet B can be used to administerthe MEK inhibitor (e.g., as a third, separate, and sequentialadministration of an active). These formulations may be prepared byconventional procedures well known in the pharmaceutical art. It will beappreciated that the above pharmaceutical compositions may be variedaccording to well-known pharmaceutical techniques to accommodatediffering amounts and types of the active agents. Additionally, thespecific ingredients and proportions are for illustrative purposes.Ingredients may be exchanged for suitable equivalents (e.g., componentsdescribed above) and proportions may be varied, according to the desiredproperties of the dosage form of interest.

While specific embodiments have been described above with reference tothe disclosed embodiments and examples, such embodiments are onlyillustrative and do not limit the scope of the invention. Changes andmodifications can be made in accordance with ordinary skill in the artwithout departing from the invention in its broader aspects as definedin the following claims.

All publications, patents, and patent documents are incorporated byreference herein, as though individually incorporated by reference. Nolimitations inconsistent with this disclosure are to be understoodtherefrom. The invention has been described with reference to variousspecific and preferred embodiments and techniques. However, it should beunderstood that many variations and modifications may be made whileremaining within the spirit and scope of the invention.

What is claimed is:
 1. A method of treating a cancer in a patient inneed thereof comprising administering to a patient, concurrently orsequentially, a therapeutically effective amount of the compound PAC-1:

and an effective amount of a second active agent, wherein the secondactive agent is an inhibitor of the BRAF enzyme having a mutation,wherein the cancer is thereby treated.
 2. The method of claim 1 whereinthe second active agent is an inhibitor of the BRAF enzyme that has theV600E or the V600K mutation.
 3. The method of claim 1 wherein the secondactive agent is vemurafenib, dabrafenib, BMS-908662, encorafenib,PLX3603, or RAF265.
 4. The method of claim 1 wherein the compound PAC-1and the second active agent are administered concurrently.
 5. The methodof claim 1 wherein the compound PAC-1 and the second active agent areadministered sequentially.
 6. The method of claim 5 wherein the compoundPAC-1 is administered before the second active agent.
 7. The method ofclaim 5 wherein the compound PAC-1 is administered after the secondactive agent.
 8. The method of claim 1 wherein the cancer is melanoma,leukemia, colorectal cancer, thyroid cancer, lung cancer, ovariancancer, Erdheim-Chester disease (ECD), or Langerhans'-cell histiocytosis(LCH).
 9. The method of claim 1 further comprising administering to thepatient, concurrently or sequentially, a therapeutically effectiveamount of a MEK inhibitor.
 10. The method of claim 9 wherein the MEKinhibitor is trametinib, cobimetinib, binimetinib (MEK162), selumetinib,PD-325901, CI-1040, PD035901, TAK-733, or a combination thereof.
 11. Amethod of inhibiting the growth or proliferation of cancer cellscomprising contacting cancer cells with an effective amount of thecompound PAC-1:

and an effective amount of a second active agent, wherein the secondactive agent is an inhibitor of the BRAF enzyme having a mutation,thereby inhibiting the growth or proliferation of the cancer cells. 12.The method of claim 11 wherein the cancer cells are melanoma cells,lymphoma cells, leukemia cells, osteosarcoma cells, breast cancer cells,or ovarian carcinoma cells.
 13. A method of inducing apoptosis in acancer cell comprising contacting the cancer cell with an effectiveamount of the compound PAC-1:

and an effective amount of a second active agent, wherein the secondactive agent is an inhibitor of the BRAF enzyme having a mutation,wherein apoptosis is thereby induced in the cancer cell.
 14. The methodof claim 13 wherein the second active agent is an inhibitor of the BRAFenzyme that has the V600E or the V600K mutation.
 15. The method of claim13 wherein the second active agent is vemurafenib, dabrafenib,BMS-908662, encorafenib, PLX3603, or RAF265.
 16. The method of claim 13further comprising contacting the cancer cell with an effective amountof a MEK inhibitor.
 17. The method of claim 13 wherein the contacting isin vivo.
 18. The method of claim 13 wherein the cancer cell is contactedwith PAC-1 and the second active agent concurrently.
 19. The method ofclaim 13 wherein the cancer cell is contacted with PAC-1 prior tocontacting the cancer cell with the second active agent.
 20. The methodof claim 13 wherein the cancer cell is contacted with PAC-1 aftercontacting the cancer cell with the second active agent.